Biochimica et Biophysica Acta 1473 (1999) 108^122

www.elsevier.com/locate/bba

Review
Physiological aspects of chitin catabolism in marine bacteria1
2
Nemat O. Keyhani *, Saul Roseman
Department of Biology and the McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, MD 21218, USA

Received 31 May 1999; received in revised form 2 August 1999; accepted 4 August 1999

Abstract

Chitin, a carbohydrate polymer composed of alternating L-1,4-linked N-acetylglucosamine residues is the second most
abundant organic compound in nature. In the aquatic biosphere alone, it is estimated that more than 1011 metric tons of
chitin are produced annually. If this enormous quantity of insoluble carbon and nitrogen was not converted to biologically
useful material, the oceans would be depleted of these elements in a matter of decades. In fact, marine sediments contain only
traces of chitin, and the turnover of the polysaccharide is attributed primarily to marine bacteria, but the overall process
involves many steps, most of which remain to be elucidated. Marine bacteria possess complex signal transduction systems
for: (1) finding chitin, (2) adhering to chitinaceous substrata, (3) degrading the chitin to oligosaccharides, (4) transporting the
oligosaccharides to the cytoplasm, and (5) catabolizing the transport products to fructose-6-P, acetate and NH3 . The proteins
and enzymes are located extracellularly, in the cell envelope, the periplasmic space, the inner membrane and the cytoplasm.
In addition to these levels of complexity, the various components of these systems appear to be carefully coordinated by
intricate regulatory mechanisms. ß 1999 Elsevier Science B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

2. Sensing and moving towards the food source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

3. Adhesion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

4. The enzymology of chitin degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

5. Regulation of the chitin catabolic cascade . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

6. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

* Corresponding author..
1
This paper is publication 521 from the McCollum-Pratt Institute.
2
It is a great pleasure to dedicate this article to two close friends and two outstanding pioneers in glycobiology, Harry Schachter and
Akira Kobata. Harry taught us all about the intricacies of glycosyltransferases and what they mean to cell surfaces, and also desperately
(and despairingly) tried to teach me enzyme kinetics. Akira showed us that there is an underlying unity and theme in the apparent chaos
of glycoprotein structures.

0304-4165 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 1 6 5 ( 9 9 ) 0 0 1 7 2 - 5

BBAGEN 24920 17-11-99

 N.O. Keyhani, S. Roseman / Biochimica et Biophysica Acta 1473 (1999) 108^122
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
Chitin, a highly insoluble biopolymer, is one of the
most abundant organic compounds in nature. The
polysaccharide is composed of linear chains of L-
(1,4)-linked N-acetylglucosamine (GlcNAc) residues
that are highly cross-linked by hydrogen bonds, sim-
ilar to cellulose. There is a growing literature on the
uses of chitin and chitosan (deacetylated chitin) and
their derivatives for biomedical, agricultural and
even cosmetic purposes [1,2]. These polysaccharides
are distributed throughout all kingdoms, Fungi,
Plantae and Animalia [3^5]. Chitin is a crucial com-
ponent of the cell walls of molds, mushrooms, cer-
tain green algae and is a major constituent of the
cuticles and exoskeletons of worms, mollusks and
arthropods. Chemically detectable chitin has been
found in 25 million year old fossilized insects [6].
Chitin production in the aquatic biosphere is enor-
mous; estimates of the annual quantity of chitin gen-
erated by a single genus of marine zooplankton (co-
pepods) exceeds billions of tons [5,7]. Dynamic
processes such as molting of cuticles as well as sen-
escence results in a continuous rain of chitin to the
ocean £oors known as `marine snow' [8,9]. Despite
this continuous deposition of a highly insoluble pol-
ymer, ocean sediments contain only traces of chitin.
This apparent anomaly was resolved by the discovery
that chitinolytic bacteria are ubiquitous in the marine
environment [10] and it is a¤rmed that these micro-
organisms are responsible for a signi¢cant portion,
perhaps the bulk, of the chitin recycling process.
Measurements on the dissolution of crab shells im-
mersed in sea water, for example, showed that the
half-life of a shell is approximately 2 weeks [11].
The hydrolysis of chitin to GlcNAc and GlcNAc
oligosaccharides has been studied for many decades
[12^15]. The glycosidases are distributed in organ-
isms ranging from bacteria and plants to man [14^
17]. Many recent reviews have covered the classi¢ca-
tion, distribution, molecular biology and structures
of various chitinases [18^21], but there are few re-
ports on the physiological processes that mediate
BBAGEN 24920 17-11-99
109
120
120
chitin catabolism in chitinolytic organisms such as
marine bacteria. The present review will focus on
these phenomena and will emphasize results obtained
with one marine bacterium, Vibrio furnissii. Vibrios
are Gram-negative facultative anaerobes, closely re-
lated to Escherichia coli, and are the most abundant
microbes in the marine biosphere [22].
Chitin degradation in the marine environment
comprises at least four major steps (Fig. 1): (1) sens-
ing of chitin either by random collision or by chemo-
taxis as explained below; (2) attachment to the chitin
to stay in close proximity to the nutrient; (3) expres-
sion of a multitude of enzymes and other proteins
required for catabolism of the polymer; (4) uptake
and catabolism of the hydrolysis products of the gly-
cosidases.
2. Sensing and moving towards the food source
How does a free swimming bacterium sense an
insoluble polymer such as chitin? One possibility is
by a passive mechanism, such as random collision.
Measurements of bacterial biomass in the water col-
umn, however, indicate that only a small fraction of
microbial populations are free-living [23,24], which
reduces the probability of random collision. The
vast majority of marine bacteria are associated with
other organisms, forming predatory or commensal
communities with microalgae, protozoans, inverte-
brates such as crustaceans and even with marine ver-
tebrates [24]. The concentrations of bacteria in algae
and marine snow are two to ¢ve orders of magnitude
greater than the concentrations in the surrounding
waters [25]. Simulations on the clustering of motile
bacteria around planktonic cells in moderate to high
turbulence levels in a mixed aquatic layer indicate
that chemotactic bacteria have a signi¢cant advant-
age over non-chemotactic bacteria in exposure and
access to nutrient sources [26,27]. These analyses on
the e¡ects of chemosensory motility of bacteria to-
wards or within a cloud of `leaked material' around
planktonic algae or marine snow underscores the im-
110 N.O. Keyhani, S. Roseman / Biochimica et Biophysica Acta 1473 (1999) 108^122
Fig. 1. Chitin degradation by marine Vibrios. The drawing sum-
marizes the physiological responses involved in chitin degrada-
tion. Marine bacteria are able to: (1) sense and move towards
nutrient (chitin) sources, (2) attach to the chitinaceous substra-
tum, (3) produce a battery of enzyme for the hydrolysis and
uptake of the resultant oligosaccharides. In V. furnissii, these
processes involve at least three signal transduction pathways
and are carefully regulated.
portance of this physiological response. It seems
likely, therefore, that chitinoclastic bacteria possess
active methods for seeking the chitin, i.e., chemo-
taxis. As noted by Jackson [27]: `Chemotaxis should
enhance interactions with large marine snow aggre-
gates, both by allowing bacteria to stay for signi¢-
cant periods around them and by enhancing the rates
at which bacteria attach to the aggregate surfaces.'
Bacterial chemotaxis has been extensively studied
in E. coli and Salmonella typhimurium, and many of
the proteins, including the £agella, the chemorecep-
tors and the signal transducing pathways have been
characterized [28]. In these organisms, the chemoat-
tractant forms a complex with its receptor in the
periplasmic space or the cytoplasmic membrane,
and the signal is then transferred to the £agellar mo-
tor by a sequential series of phosphotransfer reac-
tions from one che (chemotaxis) protein to the next.
Marine bacteria such as Vibrio alginolyticus and
Vibrio parahaemolyticus have been shown to possess
two distinct motility organelles: (1) polar £agella for
swimming in an aqueous environment and (2) lateral
£agella for swarming across solid surfaces [29^31]. In
these organisms, polar £agella are constitutively ex-
pressed and use the Na‡ motive force as an energy
source whereas lateral £agella are induced by growth
BBAGEN 24920 17-11-99
on surfaces or in high-viscosity solutions and appear
to use the H‡ motive force as an energy source [32].
Expression of lateral £agella is also regulated by the
polar £agellar motor [33] and these systems may act
synergistically, mediating functionally di¡erent che-
motactic responses to either attractants or repellents
[30]. In marine bacterial systems, chemotaxis and
motility are closely linked to invasion of hosts by
pathogenic strains and to the association of bacteria
with mucosal surfaces. For example, loss of motility
resulted in a 500-fold decrease in the virulence of
Vibrio anguillarum to its ¢sh host [34]. Additionally,
V. cholerae exhibited chemotaxis to a wide variety of
compounds including most L-amino acids as well as
to digests of rabbit mucosa [35]. Although not re-
quired for the latter stages of infection, V. cholerae
chemotaxis is thought to be necessary for reaching
mucosal cell surfaces and motility may be needed for
the penetration of the mucus gel [35]. V. anguillarum
and V. alginolyticus also exhibit chemotaxis (and ad-
hesion) toward skin, gill and intestinal mucus derived
from the gilt-head sea bream [36]. It should be noted
that the motile nature of natural microbial commun-
ities may be quite di¡erent from that observed using
cultured bacteria. A bacterial isolate from one ma-
rine community was shown to form natural assem-
blages (or microswarms) that exhibited motility ac-
celeration speeds 10^20-fold greater than cultured
bacteria [37].
There have been several reports characterizing che-
motaxis to degradative products of carbohydrate
polymers such as cellulose and chitin. Chemotaxis
to cellobiose and hydrolytic products of plant or
fungal cell wall polysaccharides has been shown in
the cellulolytic bacterium Cellulomonas gelida and a
predatory Pseudomonas which degrades the fungus
Pythium debaryanum [38,39].
Insofar as chitin degradation is concerned, teleo-
logical reasoning suggests that chitin oligosacchar-
ides should play a major role in chemotaxis. Aside
from the studies described below, the only other re-
ports of chemotaxis to chitin oligosaccharides are by
human neutrophils and phagocytes [40,41]. The func-
tions of these chemotactic systems are not known,
but may be surmised since bacteria are targets of
phagocytes, and the polysaccharide component of
bacterial cell walls (peptidoglycan) is closely related
to that of chitin. Degradation of the cell wall would
 N.O. Keyhani, S. Roseman / Biochimica et Biophysica Acta 1473 (1999) 108^122 111

Fig. 2. Bacterial chemotaxis to chitin oligosaccharides in swarm plates. Soft agar plates were prepared containing the following carbon
sources in 50% bu¡ered arti¢cial sea water supplemented with NH3 and Pi. Column (A) (GlcNAc)6 at initial concentrations of 10 WM
(row 1), 50 WM (row 2), and 100 WM (row 3). Column (B) no carbon source (control, row 1), 1 mM (GlcNAc)2 (row 2), 1 mM
(GlcNAc)4 (row 3). Column (C) 10 mM lactate (row 1), 1 mM (GlcNAc)3 (row 2), and 1 mM (GlcNAc)5 (row 3). V. furnissii was in-
oculated into the center of each plate and allowed to swarm for 30^40 h at room temperature. The concentration of sugar in Column
A was too low to easily visualize the swarm rings, so in these experiments the cells were stained with methylumbelliferyl-GlcNAc
which is cleaved to GlcNAc and the £uorescent product. It should be noted that the cells do not swarm to lactate (Column C, row
1), although it is an excellent source for growth. An additional control, not shown, was to study taxis with a mutant de¢cient in the
GlcNAc transporter. The mutant did not exhibit chemotaxis to GlcNAc, as expected, but showed normal taxis to the oligosacchar-
ides.

therefore yield fragments similar or even identical to terium V. furnissii [42^44]. V. furnissii exhibited che-
chitin oligosaccharides. motaxis to all the chitin oligosaccharides tested,
The most extensively studied chemotaxis system (GlcNAc)n , n = 1^6, forming characteristic rings in
toward chitin oligomers has been in the marine bac- swarm plate assays using concentrations of the hexa-

BBAGEN 24920 17-11-99

112 N.O. Keyhani, S. Roseman / Biochimica et Biophysica Acta 1473 (1999) 108^122
saccharide as low as 10 mM (Fig. 2). Capillary as-
says quantitatively con¢rmed the swarm plate results
and showed the following: (a) In V. furnissii the
monosaccharide, GlcNAc, is one of the most potent
chemoattractants reported in bacteria. (b) Chemo-
taxis to (GlcNAc)n , n = 2^6, requires induction. (c)
V. furnissii expresses at least three inducible chemo-
receptors having overlapping speci¢cities for chitin
oligomers. (d) Induced levels of chemotaxis are stable
for at least 21 h of starvation and increased chemo-
taxis is evident at early starvation time points (2^5
h). (e) Metabolic intermediates such as lactate, pyr-
uvate, succinate and fumarate inhibit chemotaxis. (f)
Of other chemoattractants tested, glucose and treha-
lose were as potent as attractants as GlcNAc. Treha-
lose is of note because it is a major carbohydrate
storage product in fungi and along with glucose
(sometimes without glucose) is the sugar found in
the hemolymph of insects and crustaceae [45,46].
Thus, the free-swimming cells in the environment
can be attracted to these organisms when they leak
soluble chitin oligosaccharides during molting or
from exudates (e.g. hemolymph), particularly from
sick or dying organisms. Chitinases (discussed below)
produced either by the bacteria or by the chitin con-
taining organism itself would be largely responsible
for the production of soluble chitin oligosaccharide
gradients.
3. Adhesion
Microbial adhesion to solid surfaces plays an im-
portant role in diverse phenomena including infec-
tion of tissues, biofouling and biodegradation. In
the marine environment, microbial adhesion may
¢ll a special requirement essential for survival. Aside
from coastal zones and sediments, marine waters are
essentially a desert for chemoorganotrophs such as
Vibrios. These waters contain no phosphate and little
to no organic nitrogen or ammonia [47]. Survival
therefore requires special adaptation. One classic ex-
ample is the observation that endemic cholera epi-
demics along the Ganges river coincide with copepod
blooms [48], which in turn coincides with V. cholerae
blooms. Only 10^20% of the bacterial population
was free swimming, the remainder was found associ-
ated with the copepods. This association explains the
BBAGEN 24920 17-11-99
resistance of V. cholerae to stomach acid, normally a
highly e¡ective barrier against infection. The organ-
ism `burrows' into copepod carapaces, where the in-
soluble matrix apparently a¡ords protection against
its microenvironment [49,50]. Furthermore, it has
been shown that certain `non-cultivatable' pathogen-
ic strains of Vibrio and Aeromonas can be resusci-
tated from the exoskeletons of aquatic arthropods
[51]. These data suggest that various species of zoo-
and phytoplankton may act as reservoirs of potential
toxigenic bacterial species. Thus, along with the sea
£oor and microorganisms in the planktonic environ-
ment, bacterial micro£ora associated on the surfaces
of other creatures probably represents one of the
major microbial environments in the sea [24,52]. Bac-
teria are capable of attaching to and degrading dis-
carded molts and the exoskeletons of dead animals
(Fig. 3). In addition, crustacean surfaces of living
creatures and their chitinous components in particu-
lar, are noted for supporting extensive bacterial at-
tachment and growth. Such epibiosis or `living-on'
relationships are increasingly regarded as playing
key roles in nutrient recycling and maintenance of
ecosystem balances [52].
Adhesion can be separated into two broad catego-
ries; speci¢c and nonspeci¢c. Speci¢c adhesion im-
plies that the bacteria produce receptor proteins with
speci¢city for particular sets of ligands, such as car-
bohydrates. Examples are also known where the li-
gand (e.g. carbohydrate) is produced by the bacte-
rium and the receptor protein is expressed by the
target cell or cell product. Speci¢c attachment often
involves ¢mbrial proteins or adhesins found at the
tips of pili which recognize and anchor the bacterium
to carbohydrate containing surfaces [53]. In some
cases, non-¢mbrial outer membrane proteins also
are known to participate in speci¢c adhesion. Non-
speci¢c adhesion can be mediated by proteins but
typically involves a variety of polymers and/or exo-
polysaccharides that result in adhesion via hydro-
phobic or ionic interactions. Several genes have
been isolated [54] that are responsible for the syn-
thesis of a polysaccharide secreted by Staphylococcus
epidermis; the polysaccharide is required for bio¢lm
formation on polystyrene and glass surfaces. Inter-
estingly, the synthesized polymer was shown to con-
sist of linear chains containing at least 130 residues
of L-1^4 linked GlcNAc.
 N.O. Keyhani, S. Roseman / Biochimica et Biophysica Acta 1473 (1999) 108^122
Most experiments on marine bacterial adhesion
have been concerned with bio¢lm formation on a
variety of substrata including glass, wood and vari-
ous polyesters, and typically have involved non-spe-
ci¢c adhesion [55]. Other types of marine adhesives
or composites have been described in organisms such
as barnacles, mussels and oysters [56]. These com-
pounds often contain ¢bers of collagen, ¢broin or
chitin dispersed in a crosslinked resin. Although the
role of bacterial exopolymers during attachment to
various substrata during bio¢lm formation is un-
clear, for purposes of this review those interactions
would be termed as non-speci¢c.
Possibly, the term `non-speci¢c' is a misnomer.
One can imagine that the formation of the bio¢lm
involves two steps: (1) deposition of an extracellular
polymer, such as a polysaccharide, on the surface;
(2) binding of the cells to the ¢rst ¢lm via cell surface
proteins such as lectins.
It should be noted, however, whether speci¢c or
not, there is growing evidence that bacteria attached
to surfaces are metabolically distinct from those in
the free-swimming state and that adhesive properties
are closely linked to availability of other nutrients
and cellular metabolism [55]. For example, studies
on a mesophilic marine Vibrio species has shown
that starvation increases (non-speci¢c) adhesiveness
to siliconized glass surfaces [57].
Although a few microbial lectins with speci¢city to
the monosaccharide GlcNAc have been reported
[53], the molecular details of bacterial attachment
to chitin has only been studied during the past few
years. Studies on a chitinolytic strain of Vibrio har-
veyi, revealed speci¢city of binding to chitin [58,59].
Detergent-extracted membranes from the organism
inhibited attachment and contained two peptides
(53 kDa and 150 kDa) which bound to chitin, but
displayed no enzymatic activities (i.e. no chitinase
activity). The 53 kDa protein was constitutively ex-
pressed whereas the 150 kDa protein was inducible
by chitin and/or chitin oligosaccharides. A 134 kDa
secreted protein termed `chitovibrin', which bound
chitin in a calcium-independent manner was reported
from V. parahemolyticus [60]. Chitovibrin expression
was induced by chitin and chitin oligosaccharides
and the puri¢ed protein displayed no apparent enzy-
matic activity, but showed strong a¤nity for chitin
and chitin-oligomers s dp 9. Chitin binding proteins
BBAGEN 24920 17-11-99
113
have also been reported in V. alginolyticus [61]. Sar-
cosyl solubilized membrane fractions contained four
peptides of 53, 35, 20, and 14 kDa, although the
quantities of the latter three proteins was lower
than the 53 kDa protein and di¡ered amongst vari-
ous strains. Expression of the V. alginolyticus pro-
teins appeared to be constitutive and no di¡erence
was observed between extracts derived from adhering
or non-adhering cells.
Studies on the adhesion of V. furnissii to carbohy-
drate derivatized beads, as analogues of polymers,
showed speci¢c attachment to GlcNAc [62,63]. Ad-
hesion of the cells to the beads was exceedingly and
speci¢cally sensitive to chitin oligosaccharides. The
latter not only inhibited the binding of the cells to
the beads, but when added to beads that contained
adherent cells, caused the cells to `deadhere'.
Perhaps the most interesting ¢nding in these stud-
ies were the requirements for adhesion. Cells in bu¡-
ered arti¢cial sea water did not bind to the beads,
whereas cells in complete growth medium (lactate for
energy, NH3 , phosphate and bu¡ered 50% arti¢cial
sea water) did adhere. Removing lactate prevented
binding. When phosphate or NH3 were omitted,
the cells at ¢rst bound to the beads, but after a brief
period they deadhered. The explanation for these
results came from studies with mutants, amino acid
auxotrophs, of V. furnissii. In complete medium, but
lacking the required amino acid, no binding was de-
tected. When the required amino acid was present,
normal adhesion resulted. However, when small
amounts of the amino acid were added, the cells
would bind, and then deadhere as the amino acid
was depleted from the medium. This process, adhe-
sion/deadhesion could be repeated many times by
repetitively adding the required amino acid.
To summarize, all required nutrients (lactate,
phosphate, NH3 , and in the case of the auxotrophs,
the necessary amino acid) were found to be necessary
to both permit and to maintain cell adhesion. The
implication is that there is a rapid turnover of lectin
activity and that lectin expression is a priority for
these cells. This adhesion/deadhesion apparatus per-
mits constant monitoring of the surrounding envi-
ronment and has been termed a `nutrient sensorium'.
It is a device for assaying the environment and de-
termining whether it can or cannot support survival
of this organism. If the microenvironment is inad-
114 N.O. Keyhani, S. Roseman / Biochimica et Biophysica Acta 1473 (1999) 108^122

BBAGEN 24920 17-11-99

 N.O. Keyhani, S. Roseman / Biochimica et Biophysica Acta 1473 (1999) 108^122 115

Fig. 3. Electron micrographs: (A) V. furnissii, showing two polar £agella and numerous very thin ¢mbriae. Cells are bound to a chitin
strip. (B) Bacterial degradation of chitin. Areas of chitin digestion resemble pits or craters. (C) Bacterial association with a marine
snow aggregate. (photograph in panel A courtesy of Dr. Charles Yu, Johns Hopkins University, photographs in panels B and C,
courtesy of Dr. Kevin Carmen, Louisiana State University)
6

equate to allow protein synthesis, the organism de-
parts in a search for a nutrient rich environment.
4. The enzymology of chitin degradation
Insofar as a bacterium in concerned, the ultimate
goal of chitin degradation is to convert the polymer,
(GlcNAc)n , to acetate, NH3 and fructose-6-P. The
last two steps of this sequence were established
many years ago [64,65] and the relevant enzymes
are found in V. furnissii [64^66]. (a) GlcNAc-6-
P+H2 OCAcetate+GlcNH2 -6-P; (b) GlcNH2 -6-
P+H2 OCNH3 +Fru-6-P. The problem is therefore
to de¢ne the pathway from chitin to GlcNAc-6-P.
It has been almost a century since the ¢rst chiti-
nase was described [67]. Since then, an extensive and
still expanding literature has developed on hexosami-
nidases, chitinases and chitosanases [12^17,20,21].
Many of the structural genes that encode these en-
zymes have been cloned and characterized, the pro-
teins over-produced, and the crystal structures of
some resolved [68^71]. Based on a re¢nement of
the crystal structure of a native chitinase (ChiA)
from Serratia marcesens to 2.3 A resolution, it has
been suggested that the substrate binding site of this
enzyme consists of a long groove composed of six
subunits [72]. Each subunit (named A^F) is capable
of binding a single sugar residue similar to the sit-
uation described for lysozyme [73]. Furthermore, the
reaction mechanism of the Serratia chitinase appears
to be analogous to lysozyme as well as most other
glycosidases, proceeding by general acid-base cataly-
sis [72].
Since 1939, the dogma in this ¢eld has held that
chitin hydrolysis requires only two enzymes, a chiti-
nase that cleaves the polysaccharide to the disacchar-
ide (GlcNAc)2 , and a L-N-acetylhexosaminidases (L-
GlcNAcidase or chitobiase), responsible for the hy-
drolysis of (GlcNAc)2 to GlcNAc. An unfortunate
consequence has been that many researchers have
categorized newly discovered enzymes into one of
these two groups, usually by similarity in amino
acid sequence. The literature describes a large num-
ber of `chitinases', although in many cases degrada-
tion of chitin itself was not even tested. Furthermore,
erroneous conclusions have been repeatedly reached
by the use of substrate analogues (discussed brie£y
below). However, it is slowly being recognized that
several di¡erent classes of these enzymes exist, with
some having overlapping speci¢cities. It is likely that
many of these enzymes play unique and important
roles in the physiology of chitin degradation in their
respective organisms. Various enzymes may, for ex-
ample, link degradation to processes such as adhe-
sion and chemotaxis by their e¡ects on the concen-
tration of substrate(s) and/or product(s) and these in
turn can act as inducers and/or repressors of the
di¡erent pathways.
The use of synthetic analogues, where GlcNAc or
chitin oligosaccharides are derivatized with nitrophe-
nol (para- or ortho-NP) or 4-methylumbelliferyl
(MUF) moieties has greatly facilitated the study of
many of these enzymes. These chromogenic and £u-
orogenic substrate analogues have been widely em-
ployed in the screening of recombinant libraries and
as substrates for puri¢cation and characterization of
many of these enzymes. Although much information
can be gained from the use of substrate analogues,
they can lead to mistaken conclusions if enzyme ac-
tivity is not measured using the natural substrates. It
has long been recognized that the activities of many
hydrolases when measured with synthetic substrate
analogues can be as much as a 1000-fold greater
than that observed with the natural substrate. Di¡er-
ent pH and temperature optima for arti¢cial versus
natural substrates have been reported for chitinases
isolated from Streptomyces [74] and Bacillus [75]. In
V. furnissii there are at least four enzymes which do
not belong to the classical chitinase/chitobiase en-
zyme de¢nitions [76^78]: (1) An enzyme, designated
a chitodextrinase, whose amino acid sequence shares
signi¢cant homology to chitinases but is essential-
ly incapable of hydrolyzing chitin. The chitodex-

BBAGEN 24920 17-11-99

116 N.O. Keyhani, S. Roseman / Biochimica et Biophysica Acta 1473 (1999) 108^122
trinase actively hydrolyzes chitin oligosaccharides,
(GlcNAc)n , n s 3. Furthermore, although this en-
zyme is capable of hydrolyzing the arti¢cial sub-
strates pNP-(GlcNAc)2 and MUF-(GlcNAc)2 , it
does not show any activity towards the natural di-
or tri-saccharides, (GlcNAc)2 and (GlcNAc)3 . (2) A
L-N-acetylhexosaminidase, which hydrolyzes pNP-
GlcNAc and MUF-GlcNAc, but which has no de-
tectable activity with chitin, chitin oligosaccharides
or any natural substrate tested. The physiological
function of this protein is not known but it is specu-
lated that it may play a role in the hydrolysis of
phenolic L-GlcNAc derivatives and in signal trans-
duction between invertebrate hosts and V. furnissii.
(3) A periplasmic L-N-acetylglucosaminidase. This
enzyme is highly speci¢c for the non-reducing termi-
nal GlcNAc in chitin oligosaccharides, and even
more interestingly, changes its speci¢city towards
one of these, the disaccharide, with a change in
pH. Thus, it hydrolyzes (GlcNAc)n , n = 2^6 at pH
5.8, but the activity with (GlcNAc)n , n = 3^6 in-
creases many-fold with increasing pH, while the ac-
tivity with (GlcNAc)2 decreases. At the pH of sea
water (8.0^8.3), the enzymatic activity towards the
disaccharide was virtually undetectable. Thus, careful
kinetic analysis of the enzymatic activity explained
the apparent stability of the disaccharide in the peri-
plasmic space and its subsequent uptake and induc-
tion e¡ects (see below). Interestingly, this enzyme has
substantial identity in its amino acid sequence to
human and mouse lysosomal L-N-acetylhexosamin-
idases, although these enzymes have a far di¡erent
substrate speci¢city and pH optimum than does the
V. furnissii periplasmic L-N-acetylhexosaminidase.
The human enzyme is defective in Tay-Sachs' dis-
ease. (4) Finally, a novel cytoplasmic enzyme, in-
capable of hydrolyzing pNP-GlcNAc or MUF-
GlcNAc, but which hydrolyzes (GlcNAc)2 . (J.K.
Park and N. Keyhani, unpublished observations)
Two key steps in the catabolism of the oligosac-
charides is how they get from the extracellular space
to the periplasmic space, and how the ¢nal hydrolysis
products in the periplasm, GlcNAc and (GlcNAc)2 ,
are transported across the cytoplasmic membrane.
While these subjects are beyond the scope of this
review, the complexity of the problem can be brie£y
summarized as follows: (1) In general, Gram-nega-
tive bacteria express two types of porins, or openings
BBAGEN 24920 17-11-99
in the cell envelope, non-speci¢c and sugar speci¢c
[79]. Non-speci¢c porins will admit a di- or perhaps a
trisaccharide to the periplasm. The speci¢c porins,
such as the one that functions in the uptake of mal-
todextrins, permit the di¡usion of much larger
oligomers. At this time, there is no published litera-
ture on whether there is or is not a porin that is
speci¢c for chitin oligomers. (2) Within the periplas-
mic space, the oligomers are hydrolyzed to the
mono- and disaccharides and these are taken up by
distinct transport systems [66,80]. The monosacchar-
ide transporter is driven by the phosphoenolpyruva-
te:glycose phosphotransferase system [44], so that
the product of translocation is GlcNAc-6-P. As
noted above, this compound is converted in two
steps to fructose-6-P, acetate and ammonia. In V.
furnissii, (GlcNAc)2 is accumulated at high levels,
unchanged, probably via an ATP binding cassette
(ABC) type transporter [80]. The cytoplasmic disac-
charide is thought to be hydrolyzed to the monosac-
charide, which is then phosphorylated by an ATP-
dependent kinase [66], giving GlcNAc-6-P, thus join-
ing the monosaccharide pathway. Interestingly, a
(GlcNAc)2 transporter and utilization operon has
also been identi¢ed in wild type strains of E. coli,
although this organism is not chitinolytic [81].
5. Regulation of the chitin catabolic cascade
The degradation of chitin is highly regulated in
marine bacteria, such as V. furnissii. One example
of this regulation is easily visualized (Fig. 4). In
this Figure, cells are plated at low density on chi-
tin/agar containing various nutrients. Expression of
the chitinase is evident by `clearing' of the chitin as it
is hydrolyzed. In rich broth (A), there is no clear
zone surrounding the very heavy colonies. In some-
what less rich broth (B), chitinase activity was ob-
served around small, but not large colonies. Similar
results were obtained in synthetic medium. In 0.5%
lactate (C), there are clear zones around some of the
colonies, and in 0.05% lactate medium (D), growth is
restricted (because of the limiting carbon), and there
are large clear zones. Finally, in the absence of all
other carbon sources except chitin (E), there is ex-
tensive secretion of chitinase although the colonies
are barely visible to the naked eye.
 N.O. Keyhani, S. Roseman / Biochimica et Biophysica Acta 1473 (1999) 108^122 117

Fig. 4. Induction of extracellular chitinase activity in V. furnissii grown on solid media. Cells were streaked on agar containing bu¡-
ered 50% arti¢cial sea water, NH3 and Pi, supplemented with the indicated carbon sources, and overlayed with chitin/agar containing
the same components. Plates contained: (A) high salt Luria Broth (LMB); (B) 2216 (rich) marine broth; (C) minimal plates contain-
ing 0.5% lactate in bu¡ered arti¢cial sea water (ASW); (D) minimal plates containing 0.05% lactate in ASW; (E) minimal ASW, chi-
tin is the only carbon source. Chitinase activity is visualized as clear zones around colonies. (photograph courtesy of C. Rowe, Johns
Hopkins University).

This simple experiment shows that expression of
the extracellular chitinase is highly catabolite re-
pressed and that cells secrete a maximum quantity
of the enzyme when they are starved for other car-
bon (and nitrogen?) sources.
Other proteins and enzymes in the chitin degrada-
tion pathway in chitinoclastic bacteria are also under
stringent control. In V. furnissii, expression of several
hexosaminidases, the transport system and chemo-
taxis are induced 50^100-fold by chitin oligosacchar-
ides but not by the monosaccharide, GlcNAc. These
systems are catabolite repressed by the addition of
other carbon sources such as glucose and mannitol, a
process possibly mediated by cAMP levels. Further-
more, analysis of chitinase de¢cient (phenotype)
Tn10 transposon mutants in V. furnissii, revealed a
class of mutants that mapped to the rpoS gene, which
is the gamma subunit of RNA polymerase responsi-
ble for expression of late log or stationary phase
genes (Dr. Xibing Li, unpublished results) and this
observation is in accord with the experiment in Fig.
4.
Studies using lacZ or green £uorescence protein
(gfp) fusions to a chitinase gene promoter in a ma-
rine Pseudoalteromonas sp., reported a 10-fold induc-
tion of the respective reporter genes by GlcNAc, and
three-fold by chitin in minimal media [82]. Repres-
sion was observed in rich media with and without
chitin, but was not detected in minimal media con-
taining glucose. The chitinase gene promoter was,
however, induced by starvation and high CO2 levels,
but not by metal ion, heat or cold shock or UV
exposure.
The mechanisms involved in catabolite (glucose)
repression of the chitinolytic pathways are poorly
understood and often vary even between closely re-
lated organisms. The glucokinase gene product
(glkA) has been shown to mediate chitinase produc-
tion in Streptomyces lividans, which is induced by
chitin and repressed in the presence of glucose [83].
In Streptomyces coelicolor, however, glkA is not re-
quired for glucose repression of chitinase production
(chi63) but instead repression is mediated by the
ccrA1 (carbon catabolite repression) gene [84,85].
Many bacteria utilize a signaling system for detect-
ing the density of the cell population in their imme-
diate micro-environment. The signaling system is
called `quorum sensing' and the signals (species spe-
ci¢c) are N-acylhomoserine lactones (AHL). Regula-
tion of chitinase expression by an AHL, N-hexanoyl-

BBAGEN 24920 17-11-99

118 N.O. Keyhani, S. Roseman / Biochimica et Biophysica Acta 1473 (1999) 108^122

L-homoserine lactone (HHL), has been shown in
Chromobacterium violaceum [86]. This bacterium
uses HHL as a signal molecule to regulate a wide
variety of gene expressions including production of
antibiotics and exoproteases. Similar signals are used
to control bioluminescence in V. ¢scheri [87]. A mu-
tant defective in HHL production was completely
de¢cient in chitinase activity when induced in the
presence of chitin. Addition of synthetic HHL re-
stored wild type chitinase production inducibility
[86].
To date there have been no reports directly char-
acterizing the (substrate) molecule(s) and their e¡ec-
tor(s) proteins responsible for induction of the vari-
ous enzymes and signaling pathways in chitin
degradation. Production of a transglycosylating en-
zyme in culture supernatants of an Alteromonas sp.,
which appeared to result in the production of
GlcNAc-LC6-GlcNAc, has been described [88].
Whether or not this molecule interacts with the tran-
scriptional regulatory machinery (as an inducer or
derepressor) remains to be determined. Thus, al-
though external chitin, GlcNAc, and/or (GlcNAc)n ,
n = 2^6, are known to induce various pathways in
many organisms, the nature of the true inducer(s)
remains to be elucidated.
There are few reports of genes that speci¢cally
regulate chitin degradation. Two putative regulatory
genes (chiD and chiE) were cloned as part of a chitin
utilization regulon of Serratia liquefaciens [89].
Transposon insertion or deletion of chiD resulted
in higher expression of chitinase activity, while inac-
tivation of chiE resulted in lowered chitinase expres-
sion. The lowered chitinase expression observed in
chiE mutants could be alleviated if the strain was
grown close to (i.e. streaked on the same agar plate)
a near neighbor. These results suggested that chiD
may encode for a repressor and chiE may be in-
volved in synthesis of an inducer.
Finally, a pleiotropic regulatory gene, reg1, was
identi¢ed in Streptomyces lividans, which controlled
both amylase and chitinase expression [90]. Chitinase
production in reg1 mutants was no longer induced
by chitin, but was also no longer catabolite repressed

Fig. 5. The chitin catabolic cascade. Overall dynamics of chitin degradation by marine bacteria (see text for explanation). C is the
soluble extracellular chitinase (see Fig. 4).

BBAGEN 24920 17-11-99

 N.O. Keyhani, S. Roseman / Biochimica et Biophysica Acta 1473 (1999) 108^122
(by glucose). The RegI protein displayed similarity to
members of the lactose(LacI)/galactose(GalR) re-
pressor family.
6. Concluding remarks
The quantity of chitin that is produced annually is
virtually beyond one's ability to conceptualize and
yet only traces are present in marine sediments.
Although other organisms are capable of digesting
chitin, it appears that the vast bulk of this highly
insoluble polymer is turned over by bacteria, at least
in marine ecosystems. The detailed molecular mech-
anisms of many of these events remain to be eluci-
dated, however, the emerging picture shows that chi-
tin degradation follows a highly sophisticated and
complex series of coordinated processes, each involv-
ing many genes and gene products. For example,
expression of the lectin involved in the binding of
V. furnissii to chitin, which also plays a key role in
nutrient sensing, is likely to be similar to other bac-
terial lectins. But expressing lectin activity is compli-
cated, as was shown in a series of elegant studies by
Normark and his associates [91]. E. coli lectins are
expressed at the tips of their pili and expression in-
volved 11 genes in the pap operon.
Assuming that V. furnissii is a paradigm for ma-
rine bacteria in general, we suppose that the entire
process of chitin degradation is triggered by the con-
tinuous search for food. This is especially important
in the marine biosphere where phosphate is non-ex-
istent except in the sediments and perhaps along the
coasts and where useful nitrogen is also exceedingly
limited. In regions of the ocean waters where other
nutrients are plentiful, chitin degradation is probably
of little consequence to such organisms (see Fig. 4).
But as the food supply or any necessary component
in the supply dwindles, chitin becomes an excellent
food source.
The steps that were discussed above are schemati-
cally illustrated in Fig. 5. One additional hypothesis
is advanced in this scheme, and this is to answer one
of the questions posed above, namely `how do these
organisms ¢nd their targets?' In the cartoon, two
cuticles are depicted lying near each other. Bacteria
BBAGEN 24920 17-11-99
119
attach to one (random collision? leaching of chitin
oligosaccharides?), grow and divide. The cuticle is
digested, and the cells, ultimately deprived of nour-
ishment, begin to starve. The production of chi-
tinases and their occurrence in the water at various
concentrations then plays a central role in locating
the next cuticle. One possibility is that `endogenous'
chitinases produced by the chitin containing organ-
ism at various life stages (i.e. during molting) will
result in the release of chitin oligosaccharides. Alter-
nately, as the bacteria starve, expression of the ex-
tracellular chitinase, designated C in the Figure, is
upregulated and chitinase is secreted. This soluble
enzyme di¡uses away from the cells and a few mol-
ecules of C collide with the second cuticle. C begins
to digest the chitin, forming a gradient of the prod-
uct (GlcNAc)2 . This gradient then attracts the starv-
ing cells attached to the original cuticle and the proc-
ess of chemotaxis ensues. Finally, a number of cells
¢nd the second cuticle and the degradative process
begins once again.
What is proposed here, in fact, is that a secreted
extracellular chitinase may have two functions, one
to provide the nutrient (GlcNAc)2 and the other to
create a chemotactic path to a new source of chitin.
This picture predicts that such organisms must ex-
press other chitinases, possibly a cell-associated chi-
tinase. Reasoning teleologically, the ideal arrange-
ment in the marine environment is likely to be a
cell associated chitinase which would yield products
that are immediately absorbed. This would be much
more e¤cient than depending on the C enzyme
which acts on chitin both near and far away, giving
di¡usible products, some or most of which are likely
to be lost to the environment. This idea suggests that
a single cell, depending on the surrounding condi-
tions can make one or more chitinases. Indeed, there
are frequent reports of multiple chitinases produced
by a single bacterium [7,14,15,92^94,20] although to
our knowledge there are no published explanations
for the physiological advantages of making more
than one enzyme.
In sum, we have only begun to appreciate the com-
plexity, sophistication, and indeed, the elegance of
the process by which marine bacteria catabolize
this simple polymer called chitin.
120 N.O. Keyhani, S. Roseman / Biochimica et Biophysica Acta 1473 (1999) 108^122
Acknowledgements
We gratefully acknowledge permission to use pub-
lished data and to cite unpublished results from:
Drs. Kevin Carmen (Louisiana State University),
Bonnie Bassler (Princeton University), Charles Yu
(Becton Dickinson Co.), Xibing Li and Jae K. Park
(Johns Hopkins University). Some of the studies re-
ported in this review were supported by Grants
GM51215 and GM38759 from the National Institute
of General Medical Sciences and by Grant
NA46RG0091 from the National Oceanic and At-
mospheric Administration to Maryland Sea Grant.
References
[1] R.A. Muzzarelli, Human enzymatic activities related to the
therapeutic administration of chitin derivatives, Cell. Mol.
Life Sci. 53 (1997) 131^140.
[2] Y. Shigemasa, S. Minami, Applications of chitin and chito-
san for biomaterials, Biotechnol. Genet. Eng. Rev. 13 (1995)
383^420.
[3] C. Jeuniaux, Chitinous structure, in: Comprehensive Bio-
chemistry, 1971, pp. 595^632.
[4] R.A.A. Muzzarelli, Chitin, Pergamon Press, Oxford, 1977,
pp. -309.
[5] G. Skjak-Braek, T. Anthosen, P. Sandford, Chitin and Chi-
tosan. Sources, Chemistry, Biochemistry, Physical Properties
and Applications, Elsevier, New York, 1988.
[6] B.A. Stankiewicz, D.E.G. Briggs, R.P. Evershed, M.B. Flan-
nery, M. Wuttke, Preservation of chitin in 25-million-year-
old fossils, Science 276 (1997) 1541^1543.
[7] R.A. Muzzarelli, Chitin Enzymology: Atec Edizioni, 1996.
[8] J. Johnstone, Conditions of Life in the Sea, Cambridge Uni-
versity Press, Cambridge, 1908.
[9] A.L. Alldredge, C.C. Gotschalk, The relative contribution of
marine snow of di¡erent origins to biological processes in
coastal waters, Cont. Shelf Res. 10 (1990) 41^58.
[10] C.E. Zobell, S.C. Rittenberg, The occurrence and character-
istics of chitinoclastic bacteria in the sea, J. Bacteriol. 35
(1937) 275^287.
[11] M. Poulicek, C. Jeuniaux, Chitin biomass in marine sedi-
ments, in: G. Skjak-Braek, T. Anthonsen, P. Sandford
(Eds.), Chitin and Chitosan, Elsevier Applied Science, Lon-
don, 1988, pp. 151^155.
[12] L. Zechmeister, G. Töth, Chromatographie der in der chi-
tinreihe wirksamen Enzyme des emulsins, Enzymologia 7
(1939) 165^169.
[13] L. Zechmeister, G. Töth, E. Vajda, Chromatographie der in
der chitinreihe wirksamen Enzyme der weinbergschnecke
(helix pomatia), Enzymologia 7 (1939) 170^175.
BBAGEN 24920 17-11-99
[14] J.P. Zakikas, Chitin, chitosan and related enzymes, Academ-
ic Press, New York, 1984.
[15] W.A. Wood, S.T. Kellog, Biomass, Part B, Lignin, Pectin
and Chitin. Methods in Enzymology, Academic Press, 1988.
[16] J. Flach, P.-E. Pilet, P. Jolle©s, What's new in chitinase re-
search?, Experientia 48 (1992) 701^716.
[17] G.H. Renkema, R.G. Boot, A.O. Muijsers, W.E. Donker-
Koopman, J.M.F.G. Aerts, Puri¢cation and characterization
of human chitotriosidase, a novel member of the chitinase
family of proteins, J. Biol. Chem. 270 (1995) 2198^2202.
[18] B. Henrissat, Glycosidase families, Biochem. Soc. Trans. 26
(1998) 153^156.
[19] B. Henrissat, G. Davies, Structural and sequence-based clas-
si¢cation of glycoside hydrolases, Curr. Opin. Struct. Biol. 7
(1997) 637^644.
[20] R. Cohen-Kupiec, I. Chet, The molecular biology of chitin
digestion, Curr. Opin. Biotechnol. 9 (1998) 270^277.
[21] F. Hamel, R. Boivin, C. Tremblay, G. Bellemare, Structural
and evolutionary relationships among chitinases of £owering
plants, J. Mol. Evol. 44 (1997) 614^624.
[22] R.R. Colwell, Vibrios in the Environment, John Wiley and
Sons, New York, 1984.
[23] M.R. Sochard, D.F. Wilson, B. Austin, R.R. Colwell, Bac-
teria associated with the surface and gut of marine copepods,
Appl. Environ. Microbiol. 37 (1979) 750^759.
[24] J.M. Seiburth, Microbial Seascapes, University Park Press,
Baltimore, MD, 1975.
[25] A.L. Alldredge, M.W. Silver, Characteristics, dynamics and
signi¢cance of marine snow, Prog. Oceanogr. 20 (1988) 41^
82.
[26] J.D. Bowen, K.D. Stolzenbach, S.W. Chisholm, Simulating
bacterial clustering around phytoplankton cells in a turbu-
lent ocean, Limnol. Oceanogr. 38 (1993) 36^51.
[27] G.A. Jackson, Simulation of bacterial attraction and adhe-
sion to falling particles in an aquatic environment, Limnol.
Oceanogr. 34 (1989) 514^530.
[28] R.M. Macnab, Flagella and Motility, in: F.C. Neidhardt
(Ed.) Escherichia coli and Salmonella typhimurium, ASM
Press, 1996, pp. 123^145.
[29] S. Ulitzur, E¡ect of temperature, salts, pH and other factors
on the development of peritrichous £agella in Vibrio algino-
lyticus, Arch. Microbiol. 104 (1975) 285^288.
[30] M. Homma, H. Oota, S. Kojima, I. Kawagishi, Y. Imae,
Chemotactic responses to an attractant and a repellent by
the polar and lateral £agellar system of Vibrio alginolyticus,
Microbiology 142 (1996) 2777^2783.
[31] N. Sar, L. McCarter, M. Simon, M. Silverman, Chemotactic
control of the two £agellar systems of Vibrio parahaemolyti-
cus, J. Bacteriol. 172 (1990) 334^341.
[32] D. Roby, A. Toppan, T.M.T. Esquerrë, Chitinases : Plant
defense proteins related to resistance against fungal patho-
gens, in: R. Muzzarelli, C. Jeuniaux, G.W. Gooday (Eds.),
Chitin in nature and technology, Plenum Press, New
York.,1986, pp. 231^233.
[33] I. Kawagishi, M. Imagawa, Y. Imae, L. McCarter, M. Hom-
ma, The sodium-driven polar £agellar motor of marine Vi-
 N.O. Keyhani, S. Roseman / Biochimica et Biophysica Acta 1473 (1999) 108^122
brio as the mechanosensor that regulates lateral £agellar ex-
pression, Mol. Microbiol. 20 (1996) 693^699.
[34] R. O'Toole, D.L. Milton, H. Wolf-Watz, Chemotactic mo-
tility is required for invasion of the host by the ¢sh pathogen
Vibrio anguillarum, Mol. Microbiol. 19 (1996) 625^637.
[35] R. Freter, P.C. O'Brien, Role of chemotaxis in the associa-
tion of motile bacteria with intestinal mucosa: chemotactic
responses of Vibrio cholerae and description of motile non-
chemotactic mutants, Infect. Immun. 34 (1981) 215^221.
[36] M.A. Bordas, M.C. Balebona, J.M. Rodriguez-Maroto, J.J.
Borrego, M.A. Morinigo, Chemotaxis of pathogenic vibrio
strains towards mucus surfaces of gilt-head sea brean (Spa-
rus aurata L.), Appl. Environ. Microbiol. 64 (1998) 1573^
1575.
[37] J.G. Mitchell, L. Pearson, S. Dillon, K. Kantalis, Natural
assemblages of marine bacteria exhibiting high-speed motil-
ity and large accelerations, Appl. Environ. Microbiol. 61
(1995) 4436^4440.
[38] W. Hsing, E. Canale-Parola, Cellobiose chemotaxis by the
cellulolytic bacterium Cellulomonas gelida, J. Bacteriol. 174
(1992) 7996^8002.
[39] I. Chet, R. Mitchell, Eological aspects of microbial chemo-
tactic behavior, Annu. Rev. Microbiol. 30 (1976) 221^239.
[40] K. Suzuki, A. Tokoro, Y. Okawa, S. Suzuki, M. Suzuki,
E¡ect of N-Acetylchito-oligosaccharides on activation of
phagocytes, Microbiol. Immunol. 30 (1986) 777^787.
[41] A. Tokoro, K. Suzuki, T. Matsumoto, T. Mikami, S. Suzu-
ki, M. Suzuki, Chemotactic response of human neutrophils
to N-acetyl chitohexaose in vitro, Microbiol. Immunol. 32
(1988) 387^395.
[42] B.L. Bassler, P.J. Gibbons, S. Roseman, Chemotaxis to chi-
tin oligosaccharides by Vibrio furnissii, a chitinivorous ma-
rine bacterium, Biochem. Biophys. Res. Commun. 161
(1989) 1172^1176.
[43] B.L. Bassler, P.J. Gibbons, C. Yu, S. Roseman, Chitin uti-
lization by marine bacteria: chemotaxis to chitin oligosac-
charides by Vibrio furnissii, J. Biol. Chem. 266 (1991) 24268^
24275.
[44] C. Yu, B.L. Bassler, S. Roseman, Chemotaxis of the marine
bacterium Vibrio furnissii to sugar substrates of the phos-
phoenolpyruvate: glycose phosphotransferase system,
J. Biol. Chem. 268 (1993) 9405^9409.
[45] M. Florkin, B.T. Scheer, Chemical Zoology, Arthropoda
Academic Press, New York, 1970, pp. 147^194.
[46] P.L. Altman, D.S. Dittmer, Blood and other body £uids,
Federation of American Societies for Experimental Biology,
Bethesda, MD, 1971, pp. 287^289
[47] F.G.W. Smith, Handbook of Marine Science, CRC Press,
Cleveland, OH, 1974.
[48] D.R. Nalin, Cholera, Copepods and Chitinase, Lancet 2
(1976) 958.
[49] D.R. Nalin, V. Daya, A. Reid, M.M. Levine, L. Cisneros,
Adsorption and growth of Vibrio cholerae on chitin, Infect.
Immun. 25 (1979) 768^770.
[50] D.W. lear, Symposium on Marine Microbiology, C.H. Op-
penheimer (Ed.), C.C. Thomas, Spring¢eld, IL, 1963, p. 608
BBAGEN 24920 17-11-99
121
[51] B.N. Shukla, D.V. Singh, S.C. Sanyal, Attachment of non-
culturable toxigenic vibrio cholerae-01 and non-01 and aero-
monas spp. to the aquatic arthropod gerris-spinolae and
plants in the river-Ganga, Varanasi, FEMS Immunol.
Med. Microbiol. 12 (1995) 113^120.
[52] K.R. Carman, F.C. Dobbs, Epibiotic microorganisms on
copepods and other marine crustaceans, Microsc. Res.
Tech. 37 (1997) 116^135.
[53] D. Mirelman, Microbial lectins and agglutinins, Wiley-Inter-
science, New York, 1986.
[54] C. Heilmann, O. Schweitzer, C. Gerke, N. Vanittanakom, D.
Mack, F. Gotz, Molecular basis of intercellular adhesion in
the bio¢lm-forming Staphylococcus epidermidis, Mol. Micro-
biol. 20 (1996) 1083^1091.
[55] K.E. Cooksey, B. Wigglesworth-Cooksey, Adhesion of bac-
teria and diatoms to surfaces in the sea: a review, Aquat.
Microb. Ecol. 9 (1995) 87^96.
[56] J.H. Waite, Marine adhesive proteins: natural composite
thermosets, Int. J. Biol. Macromol. 12 (1990) 139^144.
[57] M.P. Dawson, B.A. Humphrey, K.C. Marshall, Adhesion: a
tactic in the survival strategy of a marine vibrio during star-
vation, Curr. Microbiol. 6 (1981) 195^199.
[58] M.T. Montgomery, D.L. Kirchman, Role of chitin-binding
proteins in the speci¢c attachment of the marine bacterium
Vibrio harveyi to chitin, Appl. Environ. Microbiol. 59 (1993)
373^379.
[59] M.T. Montgomery, D.L. Kirchman, Induction of chitin-
binding proteins during the speci¢c attachment of the marine
bacterium Vibrio harveyi to chitin, Appl. Environ. Micro-
biol. 60 (1994) 4284^4288.
[60] O.S. Gildemeister, B.C.R. Zhu, R.A. Laine, Chitovibrin : a
chitin-binding lectin from Vibrio parahemolyticus, Glycocon-
jugate J. 11 (1994) 518^526.
[61] C. Pruzzo, A. Crippa, S. Bertone, L. Pane, A. Carli, Attach-
ment of Vibrio alginolyticus to chitin mediated by chitin-
binding proteins, Microbiology 142 (1996) 2181^2186.
[62] C. Yu, A.M. Lee, S. Roseman, The sugar-speci¢c adhesion/
deadhesion apparatus of the marine bacterium Vibrio furnis-
sii is a sensorium that continuously monitors nutrient levels
in the environment, Biochem. Biophys. Res. Commun. 149
(1987) 86^92.
[63] C. Yu, A.M. Lee, B.L. Bassler, S. Roseman, Chitin utiliza-
tion by marine bacteria: a physiological function for bacte-
rial adhesion to immobilized carbohydrates, J. Biol. Chem.
266 (1991) 24260^24267.
[64] D.G. Comb, S. Roseman, Glucosamine-6-phosphate deami-
nase, Biochim. Biophys. Acta 21 (1956) 193^194.
[65] D.G. Comb, S. Roseman, Glucosamine metabolism. IV.
Glucosamine-6-phosphate deaminase, J. Biol. Chem. 232
(1958) 807^827.
[66] B.L. Bassler, C. Yu, Y.C. Lee, S. Roseman, Chitin utiliza-
tion by marine bacteria: degradation and catabolism of chi-
tin oligosaccharides by Vibrio furnissii, J. Biol. Chem. 266
(1991) 24276^24286.
[67] M. Bernard, Sur la fonction fungicide des bulbes dPophry-
dëes, Am. Sci. Nat. Bot. Paris 14 (1911) 221^234.
122 N.O. Keyhani, S. Roseman / Biochimica et Biophysica Acta 1473 (1999) 108^122
[68] P.J. Hart, A.F. Monzingo, M.P. Ready, S.R. Ernst, J.D.
Robertus, Crystal structure of an endochitinase from Hor-
deum vulgare L. seeds, J. Mol. Biol. 229 (1993) 189^193.
[69] A.C. Terwisscha van Scheltinga, S. Armand, K.H. Kalk, A.
Isogai, B.X.D.B.W. Henrissat, Stereochemistry of chitin hy-
drolysis by a plant chitinase/lysozyme and X-ray structure of
a complex with allosamidin: evidence for substrate assisted
catalysis, Biochemistry 34 (1995) 15619^15623.
[70] S. Armand, H. Tomita, A. Heyraud, C. Gey, T. Watanabe,
B. Henrissat, Stereochemical course of the hydrolysis reac-
tion catalyzed by chitinases A1 and D from Bacillus circulans
WL-12, FEBS Lett. 343 (1994) 177^180.
[71] T. Watanabe, K. Kobori, K. Miyashita, T. Fujii, H. Sakai,
M. Uchida, H. Tanaka, Identi¢cation of glutamic acid 204
and aspartic acid 200 in chitinase A1 of Bacillus circulans
WL-12 as essential residues for chitinase activity, J. Biol.
Chem. 268 (1993) 18567^18572.
[72] A. Perrakis, I. Tews, Z. Dauter, A.B. Oppenheim, I. Chet,
K.S. Wilson, C.E. Vorgias, Crystal structure of a bacterial
chitinase at 2.3 A î resolution, Structure 2 (1994) 1169^1180.
[73] L.O. Ford, L.N. Johnson, A.C.T. North, D.C. Philips, R.
Tjian, Crystal structure of a lysozyme-tetrasaccharide lac-
tone complex, J. Mol. Biol. 88 (1974) 349^371.
[74] P.W. Robbins, C. Albright, B. Ben¢eld, Cloning and expres-
sion of a Streptomyces plicatus chitinase (chitinase-63) in
Escherichia coli, J. Biol. Chem. 263 (1988) 443^447.
[75] T. Takayanagi, K. Ajisaka, Y. Takiguchi, K. Shimahara,
Isolation and characterization of thermostable chitinase
from Bacillus licheniformis X-7u, Biochim. Biophys. Acta
1078 (1991) 404^410.
[76] N.O. Keyhani, S. Roseman, The chitin catabolic cascade in
the marine bacterium Vibrio furnissii: Molecular cloning,
isolation and characterization of a periplasmic chitodex-
trinase, J. Biol. Chem. 271 (1996) 33414^33424.
[77] N.O. Keyhani, S. Roseman, The chitin catabolic cascade in
the marine bacterium Vibrio furnissii: Molecular cloning,
isolation and characterization of a periplasmic L-N-Acetyl-
glucosaminidase, J. Biol. Chem. 271 (1996) 33425^33432.
[78] E. Chitlaru, S. Roseman, Molecular cloning and character-
ization of a novel L-N-acetyl-D-glucosaminidase from Vibrio
furnissii, J. Biol. Chem. 271 (1996) 33433^33439.
[79] G.E. Schulz, Porins: general to speci¢c, native to engineered
passive pores, Curr. Opin. Struct. Biol. 6 (1996) 485^490.
[80] N.O. Keyhani, L.-X. Wang, Y.C. Lee, S. Roseman, The
chitin catabolic cascade in the marine bacterium Vibrio fur-
nissii : Characterization of an N, NP-diacetylchitobiose trans-
port system, J. Biol. Chem. 271 (1996) 33409^33413.
[81] N.O. Keyhani, S. Roseman, Wild-type Escherichia coli grows
on the chitin disaccharide, N, NP-diacetylchitobiose, by ex-
pressing the cel operon, Proc. Natl. Acad. Sci. USA 94
(1997) 14367^14371.
BBAGEN 24920 17-11-99
[82] S. Techkarnjanaruk, S. Pongpattanakitshote, A.E. Good-
man, Use of a promoterless lacZ gene insertion to investigate
chitinase gene expression in the marine bacterium Pseudoal-
teromonas sp. strain S9, Appl. Environ. Microbiol. 63 (1997)
2989^2996.
[83] A. Saito, T. Fujii, T. Yoneyama, K. Miyashita, glkA is in-
volved in glucose repression of chitinase production in Strep-
tomyces lividans, J. Bacteriol. 180 (1998) 2911^2914.
[84] C. Ingram, J. Westpheling, The glucose kinase gene of Strep-
tomyces coelicolor is not required for glucose repression of
the chi63 promoter, J. Bacteriol. 177 (1995) 3587^3588.
[85] C. Ingram, I. Delic, J. Westpheling, ccrA1: A mutation in
Streptomyces coelicolor that a¡ects the control of catabolite
repression, J. Bacteriol. 177 (1995) 3579^3586.
[86] L.S. Chernin, M.K. Winson, J.M. Thompson, S. Haran,
B.W. Bycroft, I. Chet, P. Williams, G.S.A.B. Stewart, Chi-
tinolytic activity in Chromobacterium violaceum: Substrate
analysis and regulation by quorum sensing, J. Bacteriol.
180 (1998) 4435^4441.
[87] D.M. Sitnikov, J.B. Schineller, T.O. Baldwin, Transcription-
al regulation of bioluminesence genes from Vibrio ¢scheri,
Mol. Microbiol. 17 (1995) 801^812.
[88] K. Shimoda, K. Nakajima, Y. Hiratsuka, S.I. Nishimura, K.
Kurita, E¤cient preparation of L-(1C6)-(GlcNAc)(2) by en-
zymatic conversion of chitin and chito-oligosaccharides, Car-
bohydr. Polymers 29 (1996) 149^154.
[89] S. Joshi, M. Kozlowski, G. Selvaraj, V.N. Iyer, R.W. Da-
vies, Cloning of the genes of the chitin utilization regulon of
Serratia liquefaciens, J. Bacteriol. 170 (1988) 2984^2988.
[90] J. Nguyen, F. Francou, M.-J. Virolle, M. Guërineau, Amyl-
ase and chitinase genes in Streptomyces lividans are regulated
by reg1, a pleiotropic regulatory gene, J. Bacteriol. 179
(1997) 6383^6390.
[91] S.J. Hultgren, C.H. Jones, S. Normark, Bacterial adhesins
and their assembly, in: F.C.e.a. Neidhardt (Ed.), Escherichia
coli and Salmonella: Cellular and molecular biology, ASM
Press, Washington, DC, 1996, pp. 2730^2756.
[92] T. Watanabe, W. Oyanagi, K. Suzuki, K. Ohnishi, H. Ta-
naka, Structure of the gene encoding chitinase D of Bacillus
circulans WL-12 and possible homology of the enzyme of
other prokaryotic chitinases and class III plant chitinases,
J. Bacteriol. 174 (1992) 408^414.
[93] T. Watanabe, W. Oyanagi, K. Suzuki, H. Tanaka, Chitinase
system of Bacillus ciculans WL-12 and importance of chi-
tinase A1 in chitin degradation, J. Bacteriol. 172 (1990)
4017^4022.
[94] A.L. Svitil, S.M.N. Chadhain, J.A. Moore, D.L. Kirchman,
Chitin degradation proteins produced by the marine bacte-
rium vibrio harveyi growing on di¡erent forms of chitin,
Appl. Environ. Microbiol. 63 (1997) 408^413.