Previous Article | Next Article 
Applied and Environmental Microbiology, May 2001, p. 2380-2383, Vol. 67, No. 5
F. A. Janssens Laboratory of Genetics,
Katholieke Universiteit Leuven, B-3001 Heverlee,
Belgium,1 and Laboratoire de
Plasticité et Expression des Génomes Microbiens, CNRS
EP2029, CEA LRC12, Université J. Fourier, F-38041 Grenoble
Cedex 9,2 and Architecture et Fonction
des Macromolécules Biologiques, CNRS, F-13402 Marseille Cedex
20,3 France
Received 7 November 2000/Accepted 18 February 2001
The CelA Based on their amino acid sequence similarities,
Azospirillum bacteria colonize the rhizosphere of several
gramineous plants, such as rice, maize, sorgo, and wheat (17, 28,
29). The ability of Azospirillum irakense to grow on
pectins and plant-derived Identification of the celA locus.
Approximately
3,000 Escherichia coli clones from a genomic library of
A. irakense KBC1 (2) were tested for
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.5.2380-2383.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
The celA Gene, Encoding a Glycosyl
Hydrolase Family 3
-Glucosidase in Azospirillum
irakense, Is Required for Optimal Growth on
Cellobiosides
ABSTRACT
Top
Abstract
Text
References
-glucosidase of Azospirillum irakense,
belonging to glycosyl hydrolase family 3 (GHF3), preferentially
hydrolyzes cellobiose and releases glucose units from the
C3, C4, and C5 oligosaccharides. The growth of a 
celA mutant on
these cellobiosides was affected. In A. irakense, the
GHF3 -glucosidases appear to be functional alternatives for the GHF1
-glucosidases in the assimilation of -glucosides by other bacteria.
TEXT
Top
Abstract
Text
References
-Glucosidases (EC
3.2.1.21) are present in eukaryotic and prokaryotic organisms and
catalyze the hydrolysis of cellobiose and chemically related
-glucosides. The -glucosidases of cellulolytic and
noncellulolytic microorganisms are key enzymes for the assimilation of
cellobiose, the biodegradation product of cellulose, and other plant-derived -glucosides, such as arbutin and salicin. These enzymes also modulate the biological activities of different
-glucosides, such as antibiotics (18) or saponins
(15). Numerous -glucosidases have been identified for
cellulolytic and noncellulolytic bacteria; nevertheless, their role in
plant root colonization by plant growth-promoting bacteria is not
understood (14, 19, 22).
-glucosidases are classified into glycosyl hydrolase families 1 and
3 (GHF1 and GHF3, respectively) (4). The GHF3
-glucosidases exhibit a modular organization in two domains: an
N-terminal catalytic A domain and a C-terminal noncatalytic, but
essential, B domain. Among the bacterial enzymes, the AB organization
is the most frequent, while the BA and AB' (exhibiting a truncated
C-terminal domain referred to as B') types have also been described
(7, 13, 31). The functional analysis of GHF3
-glucosidases using genetic approaches has been documented for only
a few bacteria (7, 18, 31, 33).
-glucosides, such as cellobiose, arbutin,
and salicin, has been reported (2, 7, 11, 12). The
isolation and characterization of the corresponding enzymes could
reveal potential commercial applications (e.g., with respect to
specific activities, substrate specificities, stability, and so forth). Two GHF3 -glucosidases, SalA and SalB, are required for the growth of A. irakense on salicin (7). In this work, we
describe the characterization of a third GHF3 -glucosidase, CelA,
for A. irakense KBC1. The encoding celA gene is
required for optimal growth on cellobiose and cellulose-derived
oligosaccharides, emphasizing the importance of the GHF3 enzymes
in the assimilation of plant-derived -glucosides, a feature which is
usually assigned to GHF1 enzymes.
-glucosidase (with methylumbelliferyl--glucuronide [MUG] as a
substrate) and endoglucanase (with carboxymethyl cellulose as a
substrate) activities with overlay methods (7, 19).
No clone exhibiting endoglucanase activity was obtained. The
MUG-positive clones were classified into two families, based on the
restriction pattern of their cosmids with EcoRI,
HindIII, and BamHI. From each family, one
cosmid was retained for further characterization. pFAJ0650, harboring
two -glucosidases, SalA and SalB, has been previously described
(7); cosmid pFAJ0649 was analyzed in this work (Table
1). The cosmid insert DNA contained five
EcoRI restriction fragments, of 8, 5.5, 4.5, 3.2, and
1.5 kb. Each of these restriction fragments was subcloned into cloning
vector pBluescript SK(
). Clones showing MUG activity were
obtained only for the 5.5-kb fragment (pFAJ0680). The putative gene
encoding -glucosidase activity was named celA.
TABLE 1.
Strains and plasmids
Sequence analysis of the celA gene.
DNA
sequencing of pUC18/19 or SK() subclones was carried out with
an AutoRead sequencing kit (Pharmacia-LKB) and an automated sequencer
(ALF; Pharmacia-KLB). Sequence data were processed and analyzed by
using the PCGene software package (Intelligenetics). We sequenced, on
both strands, 2.5 kb of plasmid pFAJ0680 and found an open reading
frame encoding 685 amino acids with a predicted molecular mass of 73 kDa. The N-terminal region of this deduced amino acid sequence
exhibited a putative peptide signal 22 residues long:
MGALRLLGSISIVALTCGGIHA/STAIAQE (the slash indicates the postulated
cleavage site). This prediction of the amino-terminal signal sequence
was obtained with the SignalpWWW Server
(http://www.cbs.dtu.dk/services/SignalP/) (16).
-glucosidases (Table 2). The classification of glycosyl
hydrolases is available at the following address (4):
http://afmb.cnrs-mrs.fr/~pedro/CAZY/db.html. CelD of
Pseudomonas fluorescens showed a particularly high degree of
identity with CelA (43.6%), while a lower degree of identity was found
between CelD and SalA (24.2%) or SalB (20.5%) and between CelA and
SalA (23.4%) or SalB (22.9%). No match was obtained with the GHF1
-glucosidases. The CelA sequence has all the characteristics of the
AB enzymes: an N-terminal A domain with a conserved putative catalytic
aspartate residue and a C-terminal B domain showing the typical
conserved regions which are also present in the AB or AB' enzymes
(Table 2). A. irakense is the first bacterium for which
three orthologous GHF3 -glucosidases have been described.
|
Biochemical characteristics of CelA.
A CelA extract was
prepared from E. coli harboring plasmid pFAJ0680. Cells of
overnight cultures were collected by centrifugation, washed in
phosphate buffer (0.1 M
K2HPO4-KH2PO4
[pH 7.0]), and lysed by sonication. The lysates were cleared by
centrifugation, stored at 20°C, and analyzed for -glucosidase
activity. No -glucosidase activity was detected in E. coli with the SK() plasmid. Moreover, the insertion of a
Kmr cassette into the celA gene
abolished the expressed -glucosidase activity in E. coli
(see below). We also verified that a unique signal was present in the
zymogram of the crude CelA extract (data not shown). These features
suggested that the -glucosidase enzyme expressed from plasmid
pFAJ0680 is encoded by celA.
-D-glucoside (PNPG),
p-nitrophenyl--D-xyloside (PNPX), cellobiose, gentiobiose, and salicin as substrates. The colorimetric methods used have been described previously
(7). The optimal conditions retained for -glucosidase
activity were pH 7 and 45°C. The extract from E. coli/pFAJ0680 did not hydrolyze PNPX. The
Km determination revealed a greater
affinity for cellobiose than for gentiobiose or the aryl--glucosides
salicin and PNPG (Table 3). In the
presence of 10 mM salicin, gentiobiose, or PNPG, the specific activity
ranged from 10 to 35% of the optimal activity obtained with
cellobiose. The CelA extract also exhibited the capacity to hydrolyze
cellotriose, cellotetraose, and cellopentaose; nevertheless, optimal
activity was obtained with cellobiose as a substrate (Table 3). This
additional hydrolytic activity of CelA suggests an
exo-1,4--glucosidase activity (EC 3.2.1.74), which is also exhibited
by other GHF3 enzymes, such as CelD of P. fluorescens subsp.
cellulosa (20) and CdxA of Prevotella ruminicola (32). Despite this secondary activity, the
main characteristic of CelA is the hydrolysis of cellobiose, and the
enzyme can therefore be referred to as a cellobiase type. In A. irakense KBC1, the previously characterized adjacent
salA and salB genes were implied to be involved
in salicin assimilation, and their encoding -glucosidases exhibited
more efficient hydrolysis of aryl--glucosides than of cellobiose
(7). Moreover, the salCAB operon of A. irakense is specifically induced by aryl--glucosides and not by
cellobiose (25). These features suggest a specialization
of the three GHF3 -glucosidases in two different assimilatory
pathways for either the aryl--glucosides or the cellulose-derived
oligosaccharides.
|
Construction and phenotype of a celA A.
irakense mutant.
Plasmid pFAJ0680 was digested with
BssHII, blunt ended, and ligated with the blunt-ended
Kmr cassette isolated from pHP45
-Km (Table 1).
The resulting plasmid did not express -glucosidase activity in
E. coli transformants. This constructed deletion was
subcloned, as an EcoRI fragment, into vector pSUP202.
Triparental mating with the pRK2013 helper plasmid allowed the transfer
of the pSUP202 derivative into A. irakense KBC1.
Kmr A. irakense mutants were isolated
and checked by hybridization for double homologous recombination. The
constructed A. irakense mutant was named FAJ0694 (Fig.
1), and its growth on MMAB minimal medium
(30) supplemented with different sugars as sole carbon sources was compared with that of the wild type. Because only small
quantities of C3, C4, and C5
cellobiosides were available, all growth curves were monitored by use
of microplates with 200 µl of minimal medium.
|
-glucosides arbutin and salicin or on glucose or malate was
not affected. This phenotype is in agreement with the substrate
specificity of the CelA enzyme (Table 3). While A. irakense
uses these oligosaccharides as sole sources of carbon, it does not
assimilate carboxymethyl cellulose and cannot be regarded as a
truly cellulolytic bacterium. However, because the constructed
celA mutant reached the same cell density as the wild
type on cellobiosides after 24 h of culturing and because the
salCAB operon was not induced by cellobiose
(25), it is likely that as-yet-unidentified alternative
-glucosidases support the growth of A. irakense on cellobiosides.
GHF3 -glucosidases are alternatives for GHF1 -glucosidases in
A. irakense
The assimilatory pathways of
-glucosides are well characterized for E. coli
(23) and Erwinia chrysanthemi (5,
6). All the -glucosidases of these pathways belong to GHF1
(4), and their homologs have been identified for both
gram-positive and gram-negative bacteria (21). A.
irakense has been shown to utilize a -glucoside assimilatory
pathway involving at least three GHF3 -glucosidases, SalA, SalB, and
CelA. The GHF3 -glucosidases seem to be functional alternatives for
GHF1 -glucosidases in the assimilation of -glucosides, such as
salicin and cellobiose, in A. irakense. Moreover, the
organization and the regulation of the salCAB operon
also suggest that the assimilation of aryl--glucosides occurs via an
original mechanism which has been not described for other gram-positive
or gram-negative bacteria (25).
-glucosidases of this GHF3-COG2091
cluster is necessary for further predictive investigations. In
addition, because three orthologous -glucosidases, CelA, SalA, and
SalB, are present in the same bacterium, A. irakense KBC1,
the phylogenetic relationships among the members of this GHF3-COG2091
cluster must be clarified.
Nucleotide sequence accession number. The sequence obtained in this study was submitted to the GenBank database under accession number AF213463.
| |
ACKNOWLEDGMENTS |
|---|
D.F. was the recipient of a postdoctoral fellowship from Katholieke Universiteit Leuven (1996 and 1997). We acknowledge financial support from the Fund for Scientific Research-Flanders, the Flemish government (GOA-Vanderleyden), and the Ministry of Agriculture.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: F. A. Janssens Laboratory of Genetics, Katholieke Universiteit Leuven, K. Mercierlaan 92, B-3001 Heverlee, Belgium. Phone: (32) 16 32 16 31. Fax: (32) 16 32 19 66. E-mail: Jozef.vanderleyden{at}agr.kuleuven.ac.be.
| |
REFERENCES |
|---|
|
Top
Abstract
Text
References
|
|---|
| 1. |
Bause, E., and G. Legler.
1980.
Isolation and structure of a tryptic glycopeptide from the active site of -glucosidase A3 from Aspergillus wentii.
Biochim. Biophys. Acta
626:459-465[Medline].
|
| 2. |
Bekri, M. A.,
J. Desair,
V. Keijers,
P. Proost,
M. Searle-van Leeuwen,
J. Vanderleyden, and A. Vande Broek.
1999.
Azospirillum irakense produces a novel type of pectate lyase.
J. Bacteriol.
181:2440-2447 |
| 3. |
Castle, L. A.,
K. D. Smith, and R. O. Morris.
1992.
Cloning and sequencing of an Agrobacterium tumefaciens -glucosidase gene involved in modifying a vir-inducing plant signal molecule.
J. Bacteriol.
174:1478-1486 |
| 4. | Coutinho, P. M., and B. Henrissat. 1999. The modular structure of cellulases and other carbohydrate-active enzymes: an integrated database approach, p. 15-23. In K. Ohmiya, K. Hayashi, K. Sakka, Y. Kobayashi, S. Karita, and T. Kimura (ed.), Genetics, biochemistry and ecology of cellulose degradation. Uni Publishers Co., Tokyo, Japan. |
| 5. |
El Hassouni, M.,
M. Chippaux, and F. Barras.
1990.
Analysis of the Erwinia chrysanthemi genes which mediate metabolism of aromatic -glucosides.
J. Bacteriol.
172:6261-6267 |
| 6. |
El Hassouni, M.,
B. Henrissat,
M. Chippaux, and F. Barras.
1992.
Nucleotide sequence of the arb genes, which control -glucoside utilization in Erwinia chrysanthemi: comparison with the Escherichia coli sal operon and evidence for a new -glycohydrolase family including enzymes from eubacteria, archeabacteria, and humans.
J. Bacteriol.
174:765-777 |
| 7. |
Faure, D.,
J. Desair,
V. Keijers,
M. A. Bekri,
P. Proost,
B. Henrissat, and J. Vanderleyden.
1999.
Growth of Azospirillum irakense KBC1 on the aryl -glucoside salicin requires either SalA or SalB.
J. Bacteriol.
181:3003-3009 |
| 8. | Fellay, R., J. Frey, and H. Krisch. 1987. Interposon mutagenesis of soil and water bacteria: a family of DNA fragments designated for in vitro insertional mutagenesis of gram-negative bacteria. Gene 52:147-154[CrossRef][Medline]. |
| 9. |
Figurski, D. H., and D. R. Helinski.
1979.
Replication of an origin-containing derivative of plasmid RK2, dependent on a plasmid function provided in trans.
Proc. Natl. Acad. Sci. USA
76:1648-1652 |
| 10. | Friedman, A. M., S. R. Long, S. E. Brown, W. J. Buikema, and F. M. Ausubel. 1982. Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants. Gene 18:289-296[CrossRef][Medline]. |
| 11. | Khammas, K. M., E. Ageron, P. A. D. Grimont, and P. Kaiser. 1989. Azospirillum irakense sp. nov., a nitrogen-fixing bacterium associated with rice roots and rhizosphere soil. Res. Microbiol. 140:679-693[Medline]. |
| 12. | Khammas, K. M., and P. Kaiser. 1991. Characterization of a pectinolytic activity in Azospirillum irakense. Plant Soil 137:75-79[CrossRef]. |
| 13. | Lemesle-Varloot, L., B. Henrissat, C. Gaboriaud, V. Bissery, A. Morgat, and J. P. Mornon. 1990. Hydrophobic cluster analysis: procedures to derive structural and functional information from 2D-representation of protein sequences. Biochimie 72:555-574[Medline]. |
| 14. |
Mateos, P. F.,
J. I. Jiminez-Zurdo,
J. Chen,
A. S. Squartini,
S. K. Haak,
E. Martinez-Molina,
D. H. Hubbell, and F. B. Dazzo.
1992.
Cell-associated pectinolytic and cellulolytic enzymes in Rhizobium leguminosarum biovar trifolii.
Appl. Environ. Microbiol.
58:1816-1822 |
| 15. |
Morrissey, J. P., and A. E. Osbourn.
1999.
Fungal resistance to plant antibiotics as a mechanism of pathogenesis.
Microbiol. Mol. Biol. Rev.
63:708-724 |
| 16. |
Nielsen, H.,
J. Engelbrecht,
S. Brunak, and G. von Heijne.
1997.
Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites.
Protein Eng.
10:1-6 |
| 17. | Okon, Y., and J. Vanderleyden. 1997. Root-associated Azospirillum species can stimulate plants. ASM News 63:366-370. |
| 18. | Quiros, L. M., I. Aguirrezabalaga, C. Olano, C. Mendez, and J. A. Salas. 1998. Two glycosyltransferases and a glycosidase are involved in oleandomycin modification during its biosynthesis by Streptomyces antibioticus. Mol. Microbiol. 28:1177-1185[CrossRef][Medline]. |
| 19. |
Reinhold-Hurek, B.,
T. Hurek,
M. Clayssens, and M. van Montagu.
1993.
Cloning, expression in Escherichia coli, and characterization of cellulolytic enzymes of Azoarcus sp., a root-invading diazotroph.
J. Bacteriol.
175:7056-7065 |
| 20. |
Rixon, J. E.,
L. M. A. Ferreira,
A. J. Durrant,
J. I. Laurie,
G. P. Hazlewood, and H. J. Gilbert.
1992.
Characterization of the gene celD and its encoded product 1,4--D-glucan hydrolase D from Pseudomonas fluorescens subsp. cellulosa.
Biochem. J.
285:947-955.
|
| 21. | Rutberg, B. 1997. Antitermination of transcription of catabolic operons. Mol. Microbiol. 23:413-421[CrossRef][Medline]. |
| 22. | Safo-Sampah, S., and J. G. Torrey. 1988. Polysaccharide-hydrolyzing enzymes of Frankia (Actinomycetales). Plant Soil 112:89-97[CrossRef]. |
| 23. |
Schnetz, K.,
C. Toloczyki, and B. Rak.
1987.
-Glucoside (bgl) operon of Escherichia coli: nucleotide sequence, genetic organization, and possible evolutionary relationship to regulatory components of two Bacillus subtilis genes.
J. Bacteriol
169:2579-2590 |
| 24. | Simon, R., U. Priefer, and A. Pühler. 1983. A broad host range mobilisation system for in vivo genetic engineering: transposon mutagenesis in gram-negative bacteria. Bio/Technology 1:784-791[CrossRef]. |
| 25. | Somers, E., V. Keijers, M. H. Ottoy, M. Srinivasan, J. Vanderleyden, and D. Faure. 2000. The salCAB operon of Azospirillum irakense, required for growth on salicin, is negatively regulated by SalR, a transcriptional regulator of the LacI/GalR family. Mol. Gen. Genet. 263:1038-1046[CrossRef][Medline]. |
| 26. |
Staskawicz, B.,
D. Dahlbeck,
N. Keen, and C. Napoli.
1987.
Molecular characterization of cloned avirulence genes from race 0 and race 1 of Pseudomonas syringae pv. glycinea.
J. Bacteriol.
169:5789-5794 |
| 27. |
Tatusov, R. L.,
E. V. Koonin, and D. J. Lipman.
1997.
A genomic perspective on protein families.
Science
278:631-637 |
| 28. | Vande Broek, A., and J. Vanderleyden. 1995. Review: genetics of the Azospirillum-plant root association. Crit. Rev. Plant Sci. 14:445-466[CrossRef]. |
| 29. | Vande Broek, A., J. Michiels, A. Van Gool, and J. Vanderleyden. 1993. Spatial-temporal colonization patterns of Azospirillum brasilense on the wheat root surface and expression of the bacterial nifH gene during association. Mol. Plant-Microbe Interact. 6:592-600. |
| 30. |
Vanstockem, M.,
K. Michiels,
J. Vanderleyden, and A. P. Van Gool.
1987.
Transposon mutagenesis of Azospirillum brasilense and Azospirillum lipoferum: physical analysis of Tn5 and Tn5-Mob insertion mutants.
Appl. Environ. Microbiol.
53:410-415 |
| 31. |
Vroemen, S.,
J. Heldens,
C. Boyd,
B. Henrissat, and N. T. Keen.
1995.
Cloning and characterization of the bgxA gene from Erwinia chrysanthemi D1 which encodes a -glucosidase/xylosidase enzyme.
Mol. Gen. Genet.
246:465-477[CrossRef][Medline].
|
| 32. |
Wulff-Strobel, C. R., and D. B. Wilson.
1995.
Cloning, sequencing, and characterization of a membrane-associated Prevotella ruminicola B14 -glucosidase with cellodextrinase and cyanoglycosidase activities.
J. Bacteriol.
177:5884-5890 |
| 33. |
Yang, M.,
S. M. Luoh,
A. Goddard,
D. Reilly,
W. Henzel, and S. Bass.
1996.
The bglX gene located at 47.8 min on the Escherichia coli chromosome encodes a periplasmic beta-glucosidase.
Microbiology
142:1659-1665 |
| 34. | Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103-119[CrossRef][Medline]. |
Applied and Environmental Microbiology, May 2001, p. 2380-2383, Vol. 67, No. 5
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.5.2380-2383.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Dodd, D., Kocherginskaya, S. A., Spies, M. A., Beery, K. E., Abbas, C. A., Mackie, R. I., Cann, I. K. O.
(2009). Biochemical Analysis of a {beta}-D-Xylosidase and a Bifunctional Xylanase-Ferulic Acid Esterase from a Xylanolytic Gene Cluster in Prevotella ruminicola 23. J. Bacteriol.
191: 3328-3338
[Abstract] [Full Text] -
Faure, D.
(2002). The Family-3 Glycoside Hydrolases: from Housekeeping Functions to Host-Microbe Interactions. Appl. Environ. Microbiol.
68: 1485-1490
[Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»