University of Massachusetts Amherst ScholarWorks@UMass Amherst
Masters Theses 1911 - February 2014
1987
Pectic enzyme and protease production in Erwinia carotovora subsp. atroseptica /
Helga L. George University of Massachusetts Amherst
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PECTIC ENZYME AND PROTEASE PRODUCTION IN Erwinia carotovora subsp.
atroseptica
A Thesis Presented
by
HELGA L. GEORGE
Submitted to the Graduate School of the University of Massachusetts in partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE
September, 1987
Department of Plant Pathology PECTIC ENZYME AND PROTEASE PRODUCTION IN Erwinia carotovora subsp.
atroseptica
A Thesis Presented
by
HELGA L. GEORGE
Approved as to style and content by:
Dr. Mark S. Mount, Chairman of Committee
Dr. Mark S. Mount, Department Head Department of Plant Pathology ACKNOWLEDGEMENTS
I would like to acknowledge the support of many people who helped me throughout this project. First, I would like to thank my chairman, Dr. Mount, for assigning me a very interesting project and for providing suggestions and support throughout the project. I also want to thank Dr. George Agrios for serving on my committee and for his thorough editing of this thesis. Phyllis Berman helped me a lot by sharing her expertise on Erwinia and pectic enzymes.
My parents, Thomas and Carola George, are deserving of much credit for their constant support and encouragement throughout my education and career. I would probably not have made it this far without their support. My friends have also been
a great help to me. I want to thank Xiao Hua Li, Brian Monks, and Franzine Smith
for technical assistance and moral support, and I thank all of my friends for their
support throughout this project.
I am very grateful to the people who helped me with various computer systems.
Barr Ticknor introduced me to numerous features of WordPerfect and the LaserWriter,
and Patty and Dave Matthews taught me how to utilize the graphics features of the
Macintosh. Without their help and patience, it would have been much harder for me
to finish this thesis. I would also like to acknowledge George Allen for lending me
his IBM PC at a crucial juncture. I also want to thank my current advisor. Dr. Hans
Van Etten, for being patient while I finished this thesis under his tutelage. TABLE OF CONTENTS
ACKNOWLEDGEMENTS.i i i LIST OF TABLES.vi LIST OF FIGURES.vii
Chapter 1. LITERATURE REVIEW 1
Epidemiology.1 Introduction.1 Effect of Temperature on Pathogenicity of Eca.5 Pectic Enzymes.10 Plant Cell Wall Structure and Pectin.10 Types of Pectic Enzymes.13 Pectic Enzyme Production by Erwinia chrysanthemi.10 Pectic Enzyme Complex.16 Patterns of Pectic Enzyme Production.22 Pectic Enzyme Production by Erwinia carotovora subsp. carotovora.23 Pectic Enzyme Complex.23 Patterns of Pectic Enzyme Production.26 Pectic Enzyme Production by Erwinia carotovora subsp. atroseptica.27 Pectic Enzyme Regulation. 28 Catabolite Repression.29 Induction.30 Proposed Model for Pectate Lyase Regulation in Ech ... 33 Proteases.36 Plant Cell Wall Protein.36 Classes of Proteases.43 Involvement in Plant Pathogenesis.43 . Protease Inhibitors in Plants.45 Production of Proteases by Erwinia.46
2. CELLULAR LOCALIZATION AND CHARACTERIZATION OF PECTIC ENZYMES.
Introduction. 49 Methods. 51 Cultural Conditions. 51 Enzyme Extraction and Purification . . 51 Enzyme Assays. 53 Characterization of Enzymes. 54 Identification of Reaction Products . • 54 Polyacrylamide Gel Isoelectric Focusing 54 Results.. 55
IV Enzyme Purification.55 Characterization of Enzymes.61 Polyacrylamide Gel Isoelectric Focusing.67 Discussion.70
3. THE EFFECT OF TEMPERATURE ON PECTATE LYASE PRODUCTION . 79
Introduction.79 Methods.80 Cultural Conditions.80 Enzyme Extraction, Purification, and Assays.80 Polyacrylamide Gel Isoelectric Focusing.81 Results.82 Growth Curves.82 Enzyme Activity.82 Polyacrylamide Gel Isoelectric Focusing.92 Discussion.94
4. PARTIAL PURIFICATION AND CHARACTERIZATION OF EXTRACELLULAR PROTEASE.98
Introduction.98 Methods.99 Cultural Conditions.99 Enzyme Extraction and Purification.99 Enzyme Assay.99 Characterization of Enzyme.100 Polyacrylamide Gel Isoelectric Focusing.100 Results.101 Enzyme Purification.101 Effect of EDTA and Reactivation by Metal Ions.101 Polyacrylamide Gel Isoelectric Focusing.101 Discussion.107
APPENDIX.112 BIBLIOGRAPHY.113
V LIST OF TABLES
Table 1. Distribution of enzyme activities in subcellular fractions.56
Table 2. Chromatographic mobilities of products formed from the degradation of sodium polypectate by pectic enzymes.63
Table 3. Effect of divalent cations on the activity of pectate lyase enzymes ... 65
Table 4. The specific activity of pectate lyase peaks from DEAE column chromatography of Erwinia carotovora subsp. atroseptica incubated at 15°C and 30“C.89
Table 5. The units of pectate lyase activity in the subcellular fractions of Erwinia carotovora subsp. atroseptica incubated at 15°C and 30“C.90
Table 6. The distribution of pectate lyase activity in the subcellular fractions of Erwinia carotovora subsp. atroseptica incubated at 15°C and30°C.91
Table 7. Restoration of protease activity by metal ions.104
VI LIST OF FIGURES
Figure 1. Major pathways of polygalacturonate catabolism in bacteria.17
Figure 2. DEAE cellulose column chromatography of extracellula, periplasmic, and cytoplasmic pectic enzymes.57
Figure 3. Isoelectric focusing of the extracellular pectic enzymes.59
Figure 4. Isoelectric focusing of the periplasmic pectic enzymes.60
Figure 5. Isoelectric focusing of the cytoplasmic pectic enzymes.62
Figure 6. Effect of pH on activity of endo pectate lyase enzymes.64
Figure 7. Effect of pH on activity of exo pectate lyase.66
Figure 8. Effect of pH on activity of polygalacturonase enzymes.68
Figure 9. Isoelectric focusing of the extracellular, periplasmic, and cytoplasmic pectate lyase enzymes on a polyacrylamide gel.69
Figure 10. Growth of Erwinia carotovora subsp. atroseptica in a sodium polypectate minimal salts broth at 15*C and 30*C.83
Figure 11. DEAE cellulose column chromatography of extracellular, periplasmic, and cytoplasmic pectic enzymes of Erwinia carotovora subsp. atroseptica incubated at 30“ C.84
Figure 12. DEAE cellulose column chromatography of extracellular, periplasmic, and cytoplasmic pectic enzymes of Erwinia carotovora subsp. atroseptica incubated atl5“C.86
Figure 13. Isoelectric focusing on a polyacrylamide gel of extracellular, periplasmic, and cytoplasmic pectate lyase enzymes of Erwinia carotovora subsp. atroseptica incubated at 15“C and 30“C ... 93
Figure 14. DEAE cellulose chromatography of extracellular protease.102
Figure 15. Effect of EDTA on protease activity.103
Figure 16. Reactivation by Co^ ions of protease inhibited by EDTA.105
Fisure 17. Isoelectric focusing of extracellular protease on a polyacrylamide gel.186 Chapter 1
LITERATURE REVIEW
Epidemiology Introduction
Bacterial soft rot is one of the most important causes of the microbial spoilage of potatoes (174). The principal causal agents of this disease are several species of
Envinia, collectively known as the soft rot Erwinia. This group includes Erwinia carotovora subsp. carotovora (Jones) Bergey et al. (Ecc), Erwinia carotovora subsp. atroseptica (Van Hall) Dye (Eca), and Erwinia chrysanthemi Burkholder et al. (Ech).
Ecc and Ech affect a wide variety of plants in addition to potatoes, while Eca is limited almost entirely to potatoes (227).
The bacteria occur throughout the areas in which potatoes are grown. They can survive in the soil for short periods of time (66, 165, 190), are associated with the root zones of many plants (34, 72, 149, 150, 184), and have been isolated from many different bodies of water (183). These bacteria can be disseminated by some types of insects, mechanically by cultivation, and through the generation of aerosols (9, 108,
109, 111 152, 168, 194).
The bacteria invade potatoes primarily through lenticels, although they can also enter through wounds, bruises, and growth cracks (227). They live in the intercellular spaces (89), where they can remain latent for long periods of time (227, 218). Potatoes may become infected in the field (179), but often the seed potatoes used for planting are contaminated with bacteria (5, 67, 218, 219).
Infected seed potatoes may decay soon after planting, resulting in non-emergence or blanking, or remain symptomless, producing normal plants (227). If the seed tuber
1 2
rots later in the season, as it usually does, the bacteria can spread to the stems through the vascular system (227). The bacteria in stems often remain latent, but they can become active at any time during the growing season to form a rot of the stem known as blackleg.
Environmental conditions determine which organism is associated with blackleg.
In cool regions, blackleg is caused primarily by Eca^ although both Eca and Ecc can be present on seed tubers produced by the crop (105, 172, 225). In warmer regions, both
Ecc and Eca can cause blackleg, and, in even warmer areas, Ecc and Ech may predominate (116).
Many factors affect the severity of blackleg. Large tubers produce more post¬ emergence blackleg than small tubers (227), presumably because larger tubers decay more slowly and allow sprouts to emerge from the soil before they are invaded.
Typical blackleg expression is favored by cool moist conditions in the period immediately after planting, followed by warm moist conditions after plant emergence.
Soil moisture may gready influence the progress of the disease; stem rot usually occurs under wet conditions (221).
The size of the Eca populations in the tuber and stem greatly affect the progress of the disease. A progressive lesion will develop only when the bacterial numbers have reached a critical level, usually about 10® cells per site (222). High inoculum densities in the tuber result in seedpiece decay, while lower inoculum densities do not cause
disease initially but may result in stem decay later in the season (193). For stem rot
to occur, the populations of bacteria need to reach certain levels (80, 216). In the variety Majestic, a threshold population of about 10® bacteria per gram of stem was considered to be a prerequisite for stem decay (80).
When older plants are affected with blackleg, the bacteria can migrate along 3
underground stems into the heel ends of newly forming tubers (104, 107). These tubers
are usually able to resist infection by forming a periderm, but they still contain latent populations of bacteria. When potatoes infected in this manner rot, the disease is referred to as stolon end rot (222).
Plants may also become infected throughout the growing season through stem
wounds, and stem lesions can serve as a continuing source of inoculum. Infections
limited to the stems are usually referred to as aerial stem rot (228) and can be very
severe in fields subjected to frequent overhead irrigation during periods of high
temperature (232).
Traditionally, blackleg has been considered to be a separate disease from tuber
soft rot. Eca was originally thought to be the sole causal agent of blackleg (227), but
Ecc and Ech have since been found to cause blackleg under field conditions (68, 192,
193, 216, 232, 233, 240, 273, 281, 284). Blackleg is invariably associated with tuber rot,
and it has been proposed that the term be used to include seedpiece decay, stem decay,
and postharvest tuber soft rot (228, 272).
Tubers that show no signs of infection when harvested often develop soft rot
during transit or storage (post-harvest tuber soft rot). Symptomless potatoes often
contain bacteria in lenticels or in cracks on the surface, or they may contain bacteria
internally (63, 210, 227, 270). The treatment of harvested potatoes often encourages
the development of soft rot. Potatoes are frequently injured during harvesting and
transport, and are usually subjected to a period of "curing" to promote the healing of
wounds (174). This process involves a two week treatment of high humidity and
temperatures of 13-15*C; both conditions favor the development of soft rot. If the
crop is thought to be infected, the curing period is omitted, but soft rot may still
develop at some point. 4
If a few of the potatoes in storage rot, the whole pile may quickly decay, causing large losses. Many factors affect the susceptibility of tubers in transit or storage to soft rot; most of these are also important for the development of soft rot in the field.
The level of oxygen in the atmosphere and inside the potato is thought to be the critical factor affecting the initiation of soft rot (174, 222). Under aerobic conditions,
about 10^ to 10^ cells are required to cause a lesion at temperatures under 30’C (69),
while under anaerobic conditions, less than 10^ cells are required to initiate a lesion
(69, 218, 229).
The level of oxygen is affected by many things, including temperature, relative
humidity, the presence of free moisture, and the composition of gases in the
atmosphere (174). For instance, during high relative humidity, a film of water forms
around the tuber, resulting in depletion of oxygen inside the tuber and drastically
increasing the susceptibility to soft rot (227). The effect of oxygen is on the
resistance mechanisms of the tuber, particularly suberization and periderm formation.
Oxidation of phenols to quinones has also been implicated in resistance (173). In
addition, the water potential of potatoes also affects their susceptibility to soft rot.
This may be due to effects of the water potential on the activity of pectic enzymes (1)
or to variations in the degree of esterification of the pectic polymers (305).
As mentioned previously, the bacteria need to build up large populations to be
able to initiate soft rot, and a source of nutrients must be available for this (222).
Even under conditions otherwise favorable for soft rot, the disease will not develop
unless there is an adequate supply of nutrients. Some nutrients are present in the
intercellular spaces (151), and an additional effect of oxygen depletion may be to cause
the cell membranes to leak water and solutes from the turgid potato cells (229).
Wounding of tissues also releases nutrients, and the type of injury has a great 5
effect on the ability of the tuber to respond to it. The resistance mechanisms of a tuber respond effectively to shallow cuts, but bruises are very susceptible to infection.
When the tissue is bruised, many cells are killed, and the tissue is totally altered, producing an environment conducive to bacterial multiplication (A. Kelman).
The rate of multiplication of the bacteria is particularly important for disease progression under aerobic conditions. Under these conditions, the bacteria must initiate maceration early to overcome the wound healing reactions of the tuber, which take about 48 hours to occur (212). High populations of bacteria were able to cause maceration in time to avoid containment by the wound healing reaction, while lower populations were successfully contained by the host’s defense reaction. Weber and
Wagoner (306) found a direct relationship between the growth rate of Eca strains and their virulence. The motility of the bacteria was also found to be a factor in virulence; more motile bacteria could penetrate the tuber and initiate maceration more effectively.
Effect of Temperature on Pathogenicity of Eca
Different strains of the soft rot Erwinia grow at varying temperatures. Eca is able to grow at much lower temperatures than either Ecc or Ech. Eca grows optimally
at 27'C, with a minimum at 3'C and a maximum at 35*C (227). Ecc and Ech both have
a minimum temperature of 6”C, while they have optimal temperatures of 28-30 and 34-
37, respectively. Ecc will not grow above 37-42’C, while Ech can grow above 45"C.
Most commercial potato seed stocks contain more than one type of Erwinia, and
temperature is probably the main factor that determines which organism predominates
in a given region (222). Since Eca can grow at cooler temperatures than the other
soft rot Erwinia, it predominates in cool soils. Eca was the predominant organism
isolated from seed potato stocks in Scotland (218) and is considered to be the most 6
important pathological cause of primary soft rotting of stored potatoes under UK conditions (30).
Eca is more aggressive than Ecc at rotting seed tubers at 12*C (312) and inducing decay of inoculated tubers at 16“C (226), while Ecc predominated over Eca at 22“C.
Eca caused significantly more disease in inoculated tubers than Ecc in cool soils: soils
in which the minimum temperature was 7.0-18.5“C, and the maximum temperature was
16-20"C during the first thirty days after planting (193). In warm soils, in which the
average minimum temperature was 21.4-24*C and the maximum was 29.6-35‘’C, Ecc
produced severe disease while Eca did not.
In warmer climates, Eca predominates early in the growing season (165, 312). In
Israel, mother tubers were inoculated with Eca, Ecc, and Ech in different combinations
(222). Eca predominated early potatoes planted early in the spring when the weekly
mean maximum soil temperature at 10 cm was less than 25 C. Later in the season
when the soil temperature was higher, Ecc and Ech were more abundant. The reverse
was true for potatoes planted in the autumn when the soil temperature was greater
than 25‘’C early in the season and fell to 25*0 later.
A similar situation exists for blackleg; usually the Erwinia which is most likely to
affect the stem is also the one that has outgrown the others in the rotting mother
tuber (222). Graham and Dowson (106) found that all of their isolates that induced
blackleg at temperatures below 19“C corresponded to Eca, while most of those that
caused blackleg at 25“C or higher corresponded to Ecc or Ech. Eca produced blackleg
symptoms when potato stems were inoculated at 20“C, but not at 30 C (91), and was
the predominant organism associated with stem infections in Colorado, Scotland, and
Ireland (110, 130, 192). In Oregon, Eca causes stem rot mostly early in the season (in June), while Ecc 7
becomes dominant later (in July-August) (232). In Colorado, Ecc was recovered more often when crops were planted late, when soil temperatures were higher and in warmer parts of the state (193). At sites with intermediate soil temperatures, both were recovered with equal frequency. In hot desert or semidesert regions such as parts of
Arizona or southwestern Australia, where the soil temperature is over 35"C, Ecc predominates (61, 273).
Temperature also has a great effect in determining the success of Ecc and Eca transmission by insects and the relative persistence of the two organisms in plant tissues after transmission. Fruit flies were used to transmit bacteria from vegetative material inoculated with equal proportions of Ecc and Eca to uninoculated potato plants and from inoculated potato plants to sterile vegetative material (154). At 15*C, the insects transmitted Ecc and Eca with approximately equal frequencies over an 18 hr period, while at 27 “C, the proportion of Ecc transmitted was significantly greater than
than that of Eca.
Eca is clearly better able than Ecc to cause disease at cooler temperatures, but it
is not clear whether Eca causes more severe disease at cool or moderate temperatures.
Since the incidence of stem decay is greater at cool temperatures, it has been assumed
that the total amount of disease is greater at cool temperatures. Recent research
indicates that this may not be the case (5).
Aleck and Harrison (5) have reported that Eca rots seed tubers more effectively
at warmer temperatures. Few of the tubers survive to produce plants, and with fewer
plants, there is less visible blackleg, although the total amount of disease is greater.
At lower temperatures, the bacterium is less effective at rotting tubers in the ground.
The tubers survive to produce a lot of plants, and the incidence of visible blackleg is
much greater. 8
However, other work indicates that seedpiece decay is greater at cooler temperatures and decreases as temperatures get warmer (193). The epidemiology of blackleg is complicated by the effects of soil moisture, and it is impossible to distinguish the effect of soil temperature on blackleg from that of soil moisture.
Studies of the effect of temperature on the pathogenicity of different Erwinia
strains are complicated by competition which may occur between the different strains.
Serologically distinct strains of Eca and Ecc were coinoculated into potatoes and then
monitored with antisera (65). Eca generally doubled in population at the same rate
whether it was inoculated alone or together with the Ecc strain. However, the two
Ecc strains were inhibited in the presence of Eca. Of the two Ecc strains, one was
stimulated and the other inhibited, especially at 15“C. Thus, the behavior of strains
in the plant cannot always be predicted from in vivo growth characteristics.
If Eca does cause a greater incidence of disease at lower temperatures, there may
be several reasons for it. Perombelon and Ghanekar (224) have reported that pectic
enzyme production by several strains of Eca was high at 15*C and low or absent at
30“C. They suggested that this differential production of pectic enzymes may be
responsible for the prevalence of disease at lower temperatures.
The host’s physiology is greatly affected by temperature. Lower temperatures may
increase the susceptibility of the potato to infection and increase the expression of
disease by latent populations of bacteria which are already present. Early in the
growing season, the potato mobilizes its starch reserves to provide substrates which
can be transported and used to support sprout growth (196). The starch is hydrolyzed
to the reducing sugars glucose and fructose which are then converted to sucrose and
transported to the growing sprouts (295). Sprout growth is optimal at 20-25 C (76),
while at lower temperatures, growth is slowed and sugars accumulate (196). 9
Tubers in storage that have high levels of reducing sugars show a greater susceptibility to decay by Eca and Ech (126, 214, 309), although infection through lenticels is not affected (214). Tubers which had been planted for eight weeks had the highest levels of reducing sugars and the greatest incidence of soft rot decay by
Eca (214). Plants growing under cool temperatures and accumulating large amounts of reducing sugars may be more susceptible to rot by Eca than plants which are growing more quickly under warmer temperatures.
The resistance mechanisms of the potato to disease are also affected by temperature. Suberization and periderm formation limit the entry of Eca into fresh wounds (89), and these processes occur more rapidly at higher temperatures (11, 89).
At 20“ C, tubers are protected against decay by Eca and Ecc by two days of wound healing (212), but the process of periderm formation is slower at 15“C than at 2VC
(11). At lower temperatures, wounds remain susceptible to invasion by Eca for longer periods of time, and the incidence of soft rot decay is increased.
The effect of phytoalexins on Eca is also affected by temperature. Rishitin has
been correlated with resistance to Eca (177), and, in vitro, rishitin is much more toxic
to Eca at 30“C than at 15"C (176). However, in tubers, the mean weight of rots
caused by Eca, Ecc, and Ech increased as the temperature increased from 15“C to
22“C, and from 22“C to 30“C (175). 10
Pectic Enzymes
Plant Cell Wall Structure and Pectin
Plant cell walls are complex structures that enclose the protoplasts of higher plant cells. These walls contain cellulose microfibrils embedded in a gel-like matrix, which allows the apoplastic movement of water, ions, and small molecules into the wall, and protects the protoplast from osmotic and mechanical damage. In addition, the matrix also contains a variety of hydrolytic and oxidative enzymes involved in cell
wall metabolism.
The walls which are laid down by dividing cells are known as primary cell walls.
They remain thin as the cell continues to grow and have relatively few cellulose
microfibrils (73). As the cell matures, it may deposit additional wall components on
the inner surface of the primary wall. These components comprise the secondary wall
and include additional cellulose, hemicellulose, and sometimes lignin. Secondary walls
are of particular importance in woody tissue and in cells specialized for conducting
water. Most mature cells involved with metabolic processes such as photosynthesis,
respiration, or secretion contain only primary walls (242). The region where the walls
of individual cells come together is referred to as the middle lamella.
The polysaccharides of the cell wall matrix are classified by their ease of
extraction. Those that are easily extracted by hot water, ammonium oxalate, or
chelating agents are referred to as the pectic substances, while those that require
alkali to be solubilized are known as hemicellulose (62). The remaining residue is
composed mainly of cellulose.
The pectic polymers comprise about 35% of the primary cell walls of dicots and
are a very complex group of polysaccharides. Their characteristic component is D-
galacturonic acid, which forms a backbone of a-1,4 linked D-galacturonic acid, known 11
as polygalacturonic acid (PGA). The polymers frequently contain rhamnosyl, arabinosyl, and galactosyl residues, and the pectic polysaccharides are currently considered to be "those polymers found composed mainly of galacturonosylic residues, together with covalently or non-covalently bound neutral fractions" (73).
The primary backbone of the pectic polymers is rhamnogalacturonan: PGA interspersed with rhamnose at intervals in the chain and additional side chains of various neutral sugars (78). Two types of rhamnogalacturonan are found within primary cell walls: rhamnogalacturonan I (Rgl) and rhamnogalacturonan n (Rgll).
Rgl is a large polymer that contains covalently bound side chains of arabinan and galactan. Rgll is a much smaller, highly branched polymer that includes some unusual sugars: 2-0-methyl-D-xylose, 2-0-methyl-L-fucose, apiose, aceric acid, and ketodeoxyoctonate (KDO) (12). In addition, rhamnogalacturonan has always been assumed to be attached to, or to contain, sections of unbranched PGA known as homogalacturonan. The pectic fractions of dicots also include arabinan, galactan, and they may contain arabinogalactan (73).
The various pectic polymers can be modified in different ways. The uronide moieties may be acetylated at positions 2 and 3, and there are regions in which the carboxyl groups of D-galacturonic acid are highly methylated (62). Regions which are not methyl-esterified can be cross-linked by Ca"^ ions which chelate the oxygen atoms of adjacent galacturonosyl residues. In addition, the polymers sometimes contain phenolic groups which may be cross-linked by oxidative coupling (96). There is some evidence that pectins contain ferulate groups that can be cross-linked by peroxidase (95).
The current model of the primary cell wall of dicots was proposed by Albersheim and colleagues (2). Cellulose microfibrils are covered by a layer of hemicellulose. 12
which is interconnected by the pectic polysaccharides (62). It was proposed that the cellulose is coated with xyloglucan, which is hydrogen-bonded to the glucose chains in the cellulose fiber. The other end of the xyloglucan is glycosidically bonded to an arabinogalactan molecule. The arabinogalactan is in turn bound to rhamnose in the rhamnogalacturonan chain. Each rhamnogalacturonan chain can receive several of the
arabinogalactan molecules, each radiating from different cellulose fibrils.
Hydroxyproline-rich glycoprotein (HRGP) was thought to be involved in cross-linking
by binding to arabinogalactan, however this has since been found not to be the case
(73, 187). The involvement of the HRGP was inferred from the structure of the
secreted HRGP, which has since been found to be different from the insoluble HRGP
(extensin) (231).
The involvement of HRGP in the cell wall is not clear, however it is thought to
interact noncovalently with the other cell wall constituents. Lamport and Epstein
(162) have proposed a model of primary cell wall structure in which cellulose
microfibrils coated with xyloglucan penetrate the pores of a network of extensin
molecules; there appears to be a strong association between protein and cellulose
microfibrils (81, 195). In addition, it is suspected that an extensin-pectin network
may be part of the primary cell waU structure in dicots (56, 162). Extensin is very
basic and has a set of repeating charges that could interact with the acidic pectins.
While the exact structure of the primary cell wall is not known, it is clear that
the pectic polymers have an important structural role in cross-linking various
components of the primary cell wall. In addition, pectin is a primary component of
the middle lameUa. If the pectin is degraded, the cells are no longer held together.
The primary cell walls of the dicots that have been studied so far are all very
similar to each other, and the cell walls of potato tubers are no exception. The 13
parenchyma cells of potato tubers contain only primary walls (128) which are hard to separate from the middle lamella (138). The middle lamella of the tuber cells is composed primarily of calcium pectate (185), although they may also contain a high molecular weight protein component that does not contain hydroxyproline (137).
The pectins from the cell walls of potato tubers have been extracted and studied,
and half of the components of the cell wall are pectin. 25% is composed of cellulose,
and the rest consists of protein and hemicelluloses (128). About a quarter of the
pectin was extracted by chelating agents and was not covalently bound (139), and
about 55% of the pectin was esterified (137).
Most of the pectin is comprised of unbranched a-1,4 linked homogalacturonan
regions (137), although there are regions containing neutral sugars. Ishii (138)
isolated two fractions of rhamnogalacturonan—one that corresponded to Rgl and
another, more highly branched polymer, similar to Rgll but not containing apiose,
methyl xylose, fucose, or glucuronic acid. The dominant neutral side chains of these
compounds were 1,4 linked galactans followed by 1,5 linked arabinans; both were
covalently attached (139). Small quantities of xyloglucan were covalently attached to
pectin, although there was no firm evidence that they functioned to cross-link the
pectins with other cell wall polysaccharides (139).
Types of Pectic Enzymes
The contact between extracellular cell wall degrading enzymes (CDWE) of
pathogens and host cell walls is often one of the first molecular interactions between
host and parasite, the outcome of which can modify the type or balance of the
relationship (59). Erwinia species produce a variety of extracellular enzymes capable
of degrading components of the plant cell wall, and it is the production of these
enzymes that enables the soft rot bacteria to macerate tissue so effectively. 14
Since the turn of the century, pectic enzymes have been considered the primary agents responsible for the maceration of tissue (43). Pectic enzymes degrade the rhamnogalacturonan chains which make up the primary cell wall and middle lamella of plant cell walls, leading to cell separation and death. The initial attack by pectic enzymes allow other extracellular enzymes such as protease, cellulase, phospholipase,
and hemicellulases to degrade newly accessible sites in the plant cell wall (59).
The application of purified pectic enzymes to plant tissue can produce maceration,
but not all groups of pectic enzymes are able individually to reproduce all observed
aspects of the maceration process (43). The soft rot Erwinia produce an array of
pectic enzymes, with varying roles in pathogenesis. Pectin methylesterase (PME)
removes the ester groups from esterified pectin and is said to be important in systems
in which polygalacturonase is the primary agent of degradation (45). However, it has
not been shown to be important in bacterial soft rot pathogenesis. The important
enzymes are those that cleave the galacturonan backbone, degrading it to small
fragments that can be assimilated by the bacteria.
Two classes of enzymes degrade galacturonan in this manner. Polygalacturonase
(PG) acts hydrolytically, and pectate lyase (PL) degrades lyitically by B-elimination to
form an unsaturated bond between and of the galacturonic acid molecule.
These enzymes can be further subdivided on their mode of action. Endo enzymes
attack internal regions of the chains at random, and exo enzymes systematically
cleave terminal residues. Pectate lyases and polygalacturonases can usually be differentiated by their pH
preferences and cofactor requirements. PL’s are most active under alkaUne conditions
and have pH optimums ranging from pH 8.0 to 9.5 (246). They are stimulated by
cofactors and usually require them for activity. Polygalacturonases have pH optima 15
from pH 4.0 to 6.5 and have no activity under alkaline conditions. They do not require cofactors and are frequently inhibited by them.
EndoPG’s are specific for pectic acids, requiring free carboxyl groups for activity
(88), and prefer high molecular weight galacturonans (43). Their rate of activity
decreases as the substrate chain gets shorter (246). The primary end products of
these enzymes are mono and digalacturonic acids (88). ExoPG’s degrade
polygalacturonic acid from the non-reducing end and are usually inhibited by
unsaturated bonds (43, 246). They do not degrade pectic acid completely.
EndoPL’s can be divided into two groups, depending on their mode of action.
One group can cleave unsaturated tetramers at the central bond and can further
degrade unsaturated trimers. It produces unsaturated dimers as its major end product
(43, 246). The other group preferentially cleaves bond 3 of the trimer and pentamer
to produce unsaturated trimer as its major end product. EndoPL’s require cofactors
to maintain catalytic activity (43, 246).
ExoPL’s are specific for the penultimate bond of the reducing end of the
D-galacturonan and produce unsaturated dimer as their sole reaction product (43, 246).
Some exoPL’s can still catalyze reactions without cofactors, although they are less
effective (136, 169). The endo acting enzymes have been found to be most important for the actual
maceration of tissue. Both endoPL’s and endoPG’s have been found to macerate tissue
(59). The exo enzymes have not been found to macerate tissue, but they play
important roles in the induction of the pectic enzyme complex and wiU be discussed
later. In addition, all the strains of the soft rot Erwinia which have been examined so
far produce pectin lyase (PNL), which is specific for pectin (291). This enzyme is 16
induced by agents that damage DNA or inhibit its synthesis, and it appears to be part of the SOS system of these bacteria, the system responsible for the repair of damaged
DNA (46). PNL is often coordinately induced with bacteriocins or temperate phages.
It is not produced in response to pectic substances, and its role in soft rot pathogenesis is not clear.
The oligouronides produced by the pectic enzymes are transported into the
bacterial cells, where they are further degraded by an array of intracellular enzymes
(Figure 1). The larger uronides are broken down to unsaturated dimers by exoPL.
The unsaturated dimers are then cleaved by oligogalacturonide lyase (OGL) to form
two molecules of DTH, also known as DKI (4-deoxy-L-treo-5-hexosulose uronic acid).
OGL also cleaves saturated dimers, converting them to one molecule of DTH and one
molecule of galacturonic acid monomer. DTH is converted by an isomer to DGH, also
known as DKII (3-deoxy-D-glycero-2,5-hexodiulosonic acid 4) (49). DGH is
converted to KDG (2-keto-3 deoxygluconate) by KDG oxidoreductase (53). KDG is
phosphorylated and then cleaved to produce pyruvate and triose phosphate.
Molecules of D-galacturonic acid monomer are metabolized by a different pathway.
Uronate isomerase converts them to D-tagaturonate which is then reduced by
tagaturonate reductase and NADH to form D-altronate. This is converted to KDG by
altronate dehydrogenase.
Pectic Enzyme Production by Erwinia chrysanthemi
Pectic Enzyme Complex. Erwinia chrysanthemi produces a variety of pectic
enzymes, including pectate lyases and polygalacturonase. The profiles of this organism
have varied widely, depending on the strain used and the type of isoelectric focusing.
Original investigations showed two to four isozymes of PL. Immunological reactions
indicated that at least two of the isozymes produced by a given strain were 17
Figure 1. Major pathways of polygalacturonate catabolism in bacteria. Enzymes and genes for the catabolic steps are: (1) polygalacturonase {peh)y (2) pectate lyase ipeJ), (3) oligogalacturonide lyase (og/)* (4) uronate isomerase {uxaC)^ (5) altronate oxidoreductase (uxaB), (6) altronate hydrolyase (uxaB), (7) 4-deoxy-L-r/irg(9-5- hexoseulose uronate isomerase (kduT), (8) 2-keto-3-deoxy-D-gluconate dehydrogenase {kduD), (9) 2-keto-3-deoxygluconate kinase (kdgK), (10) 2-keto-3-deoxy-6- phosphogluconate (kdgA). The D-galacturonate pathway consists of steps 4,5, and 6; the IQDU pathway of steps 7 and 8, and the KDG pathway of steps 10 and 11. (modified from Chateijee et al. 1985). Polygalacturonate
peh
Unsaturated Saturated Digalacturonate Digalacturonate ogl i 3 II ogl 4-deoxy-L-threo-5- D-galacturonateJtU hexoseulose uronate uxaC (DKI) 1 kdul ^ 7 D-tagaturonate 3-deoxy-D-glycero- 5 I uxaB 2,5-hexodiulosonate i D-altronate (DKII)
8 kdiD
2-keto-3-deoxy-D-gluconate (KDG) 9 iI kdgK 2-keto-3-deoxy-6-phosphogluconate (KDGP) 10 i kdgA pyruvate + triose-3-phosphate 19
antigenically distinct, whereas each may be antigenically similar to the enzymes of similar pi produced by other strains (182).
Advances in technology in recent years have shown that the PL complex is more complicated than initially thought. The introduction of ultra thin layer polyacrylamide
(PAGE) isoelectric focusing with substrate overlays has allowed the detection of enzymes that had previously been undetected (51). Molecular cloning techniques have allowed the cloning of genes for several of the PL’s, indicating that the enzymes are true isozymes and not just artifacts of the isoelectric focusing process.
Many strains of Ech produce five PL isozymes-one acidic (PLa), two neutral (PLb and PLc), and two alkaline (PLd and PLe) (50). In strain 3937, these PL’s are considered to have pi’s of 4.6, 8.2, 8.5, 9.2, and 9.3. These enzymes are expressed from five different genes which are localized at two different regions in the chromosome map. One region contains the pelB and pelC genes which encode the neutral isozymes (298). The other region contains the pelA, pelD, and pelE genes, which encode the acidic and alkaline isozymes (244, 245, 298) The two regions are loosely linked to each other (298).
In addition to the five main PL’s, additional enzymes can be produced by post-translational modifications (51). These enzymes were undetected until the advent of ultra-thin layer isoelectric focusing. Collmer et al. (51) found 4 major and 8 minor bands of PL activity from strain 1237, and Bertheau et al. (24) found up to 12 bands, including 5 major bands, using strain 3937.
Ried and Collmer (250) used thin-layer isoelectric focusing to compare the PL profiles of 9 strains of EcK representing the six subdivisions or pathovars within the taxon. They found that all of the strains exhibited multiple isozymes of PL that could be classified into 3 groups: basic (pH 9-10), neutral (pH 7-8.5), and acidic (pH 20
4-5). A strong correlation was observed between subdivision classification and PL isozyme profiles.
The five primary enzymes have been purified by sucrose column isoelectric focusing and characterized. In those cases where fewer enzymes were found, it is probable that several enzymes were combined into one peak by the isoelectric focusing. The alkaline enzymes PLd and PLe that have been studied to date have all been reported to be endo enzymes. The enzymes examined include ones of pi 9.0 and
9.4 (14), 9.1 (51), 9.4 (99), and 9.1 (100). The enzyme of pi 9.1 is thought to be composed of two enzymes (51). Keen et al. (145) cloned the gene for pelE that produced PLe with a pi of 9.8.
The modes of action of the neutral enzymes are different from the alkaline PL’s.
The neutral enzymes are considered to be endo enzymes (14, 51), but they do not macerate as effectively as the alkaline enzymes (50). These enzymes have been reported to exhibit an ’’enrichment for exo activity” (99). Collmer et al. (51) found that two genes coded for a pi 8.1 enzyme, and proposed that the peak represents a mixture of PLb and PLc.
The acidic PL has not been as well characterized as the others. Garibaldi and
Bateman (99) have reported it to be intermediate in mode of action between the endo and exo enzymes. They also found that it did not macerate or kill tissue, unlike the other enzymes. This isozyme has not always been detected, and Pupillo et al. (238) were unable to detect it in culture filtrates, but could isolate it from Diejfenbachia leaves infected with Ech.
The pH optimums presented for these enzymes vary. Again, this may reflect problems of experimental protocol. If the substrates are kept too long before use, their pH can change, leading to variable results (207). Bagley and Starr (14) found 21
the pH optimum of all of their enzymes (pi 9.4, 9.0, 7.8) to be 8.5 to 9.0. Garibaldi and Bateman (99) found different pH optimums for different isozymes: isozymes of pi
9.4, 8.4, 7.9, and 4.6 had pH optimums of 8.2, 9.5,9.5, and 8.6 respectively. In another paper (98), they found the pH optimum of the enzymes of pi 9.1 and 7.9 to be 9.6. The molecular weights presented also vary; the alkaline enzymes tend to have higher MW’s than the neutral ones (50).
The role of individual isozymes in pathogenesis has been studied by examining the ability of mutants created by site-directed mutagenesis to cause disease. These mutants are exactly like the wild type, except for the lack of production of one gene product. Boeder and Collmer (257) found that PLb’ or PLc' mutants could macerate whole potato tubers as effectively as the wild-type.
Boccara et al. (28) obtained mutants deficient in PLb, PLbc, PLc, PLd, and PLe from a strain of Ech isolated from Saintpaulia, On potato slices, all of the mutants exhibited similar amounts of maceration. However, when introduced into Saintpaulia, an assay more representative of the natural course of the disease, the differences became evident.
Mutants deficient in PLb were as pathogenic as the wild type, suggesting that this enzyme is not involved in pathogenesis. PLc‘ mutants took one week longer to produce symptoms, suggesting that this enzyme plays a role in the establishment of infection. Mutants lacking PLd could not produce generalisation, the ability of the bacteria to progress in the petioles and stem to infect other leaves. This suggests an important role in pathogenesis for the PLd enzyme. PLe' mutants were avirulent: this enzyme is crucial for disease.
E. coli clones producing high levels of PLe were substantially more virulent than
Ech, providing further evidence for the importance of this enzyme in soft rot disease 22
(215). An E. coli expression vector clone producing high levels of PLe was equivalent to Ech in its ability to macerate potato tissue (147). However, studies involving pathogenicity tests of E, coli PL clones must be interpreted with caution, since many of these tests involve the introduction of extremely high numbers of bacteria onto potatoes. Further evidence for the importance of PLe in soft rot pathogenesis comes from the work of Thum and Chateijee (283). They obtained antibodies to PLb, PLc, and PLe from strain EC 16. This strain does not produce PLd. They found that PLe comprised 50-60% of the total PL in potato tubers and induced cultures. PLe also represented a large part of the total PL activity of strain B374, which does produce
PLd (245).
Most of the cloning of PL genes has been performed in E. coli, and the enzymes
are mostly retained in the periplasm (244). Recently, the pel genes have been cloned
into Myxococcus xanthus, where they are secreted to the extracellular environment
(31). The secreted PLc is modified.
In addition to multiple isozymes of PL, some strains of Ech also produce an
exopolygalacturonase during late log phase (52). The enzyme has a pi of 8.3, a pH
optimum of 6.0, and a MW of 58,000-65,000. Unlike most exoPG’s (43), it prefers
chains with unsaturated residues, attacking from the non-reducing end in a random
multichain action pattern. This enzyme releases substantial amounts of
4,5-unsaturated residues in the form of dimers. Ried and Collmer (250) used
thin-layer isoelectric focusing to examine 9 strains of Ech for this enzyme. Strains
isolated from dicot hosts produced the enzyme, while those isolated from monocot
hosts did not. The enzyme had a pi of 8.0.
Patterns of Pectic Enzyme Production. Pectate lyase is produced from the early
exponential phase of growth in Ech (10). At the end of the logarithmic growth phase, 23
there is a wave of intracellular PL activity and a dramatic increase in the level of extracellular PL (48). Most of the PL synthesized is quickly secreted and most of the
PL produced by Ech is extracellular (39,48, 282). Of the PL that remains
intracellular, most is located in the periplasm (10, 282).
Pectic Enzyme Production by Erwinia carotovora subsp. carotovora
Pectic Enzyme Complex. The initial work on the pectic enzymes of Ecc was
done by Moran, Nasuno, and Starr (198) who used column chromatography to purify
extracellular and intracellular PL from Ec. They obtained one peak which had a pH
optimum of 8.5 and was stimulated by CaCl^. The enzyme preferred long chain
substrate and produced mainly unsaturated dimer with lesser amounts of saturated
monomer and dimer and unsaturated trimer. They assumed that the peak contained
only one enzyme, but this was probably not the case. The production of saturated
oligomers suggests that there was a PG in the preparation, and there may also have
been several PL’s. These researchers (197) also purified an CXjL from the
intracellular fraction. It had a pH optimum of 7.2 and did not require CaCl^ for
activity.
Mount et al. (205) purified an extracellular PL by isoelectric focusing and
obtained an endoPL with a pi of 9.2 and a MW of 31,000 that preferred CaCl^ as a
cofactor. They assumed that only one enzyme was present, but close examination of
the graph suggests that two other peaks are present. Tseng and Mount (287) repeated
this purification and found the enzyme to have a pi of 9.4.
Stack et aL (271) purified the intracellular PL’s by isoelectric focusing and found
four different enzymes with PL activity. PD I was an endo enzyme of pi 9.4 that
preferred Ca"^ as a cofactor but could also utilize Mg'^. At pH 8.5, it produced
unsaturated trimers through hexamers. This enzyme was thought to be the same as 24
the extracellular one previously purified by Mount et al. (205).
PD n had a pi of 8.0 and exhibited exo lyase activity over a broad pH range (pH
5.5-10.0) with optimal activity at pH 8.5. It required Mn"^ for optimal activity, but could also use Mg"^ and Ca"^. It produced primarily unsaturated dimer, but at acidic pH’s, it produced traces of unsaturated trimer and exhibited limited endo activity. PD in was an exo lyase that produced unsaturated dimers and had a pi of 6.3. The enzyme had activity over the pH range of 5.5 to 10.0 with optimal activity at pH 8.5.
This enzyme did not require divalent cations for activity, but was slightly stimulated by Ca^, Mg^, and Mn^^.
The other enzyme with PL activity, PD FV, was found to be an oligogalacturonide lyase. It had a pi of 6.5, but was unstable after isoelectric focusing. It was characterized after partial purification by column chromatography. At pH 7.5-8.5, it converted unsaturated dimer and other oligouronides to predominantly unsaturated monomer. At pH 6.0, it produced predominantly D-galacturonic acid. It also exhibited limited exo lyase and exo hydrolyase activity on sodium polypectate.
Several workers have reexamined the extracellular PL of Ecc and found several enzymes. Some of these researchers used enzyme which was purified by column chromatography, but not by isoelectric focusing (122, 141, 198). These enzymes were probably mixtures of enzymes, and the characterizations may not be definitive.
Suguira et al. (279) isolated and characterized two extracellular PL’s by isoelectric focusing. The enzymes had pi’s of 10.1 and 10.7. They both preferred Ca"^ as a
cofactor and were inhibited by Mg'^ and Mn"^. Both enzymes had a pH optimum of
10.0, and neutral sugar analysis of them indicated that they both contained
carbohydrates.
Tanabe et al. (280) purified four extracellular PL’s by isoelectric focusing. The 25
enzymes were all endo PL’s with pi’s of 10.9, 10.6,10.3, and 10.0, and they were all stimulated by Cai*^. The pH optimums were 9.5,9.3, 9.7, and 9.7, respectively, and the MW’s were 33,000, 32,000, 28,000, and 28,000.
Ried and Collmer (250) used thin-layer isoelectric focusing to check the extracellular profiles of 3 strains of Ecc. Each strain produced 3 enzymes, with
approximate pi’s of 9.0, 10.0, and 10.3 (251).
Several genes for PL’s from Ecc have been cloned into E. coli and then
characterized. However, these studies must be interpreted with caution, since E. coli
has been found to modify the PL’s that have been cloned into it (269). Zink and
Chateijee (311) cloned two PL genes from Ecc 71. One produced an endoPL of
approximate pi 8.1 that produced unsaturated dimer to multimer. The other produced
an exoPL of approximately pi 8.5 that produced unsaturated dimer and trimer.
Roberts et al (254) cloned several PL’s from ECU. These enzymes were secreted
differentially by E. coli. One was found both intracellularly and extracellularly. It
was an endoPL with a pi of 9.5 that preferred Ca"^ as a cofactor. This is probably
the same enzyme that was characterized by Mount et al. (205) and Stack et al. (271).
The other enzyme was found only extracellularly. It was an endo-like PL with a pi
of 7.5. It has not been described from Ecc. The production of these enzymes is
linked, and all of the genetic information necessary for their production is contained
within a 1.5 kb region. Roberts et al. (253) also cloned an exoPL of pi 7.8 that
produced unsaturated dimers and trimers. The enzyme preferred Mn^^ as a cofactor
and had limited activity in the presence of EDTA. This enzyme is very similar to the
PD n enzyme characterized by Stack et al. (271). Lei et al. (170) cloned three linked
genes that code for Ecc PL’s. Two of the PL’s are 44 kDal proteins with pi’s of 9.4
and the third produced a 41 kDal protein with a pi of 9.1 26
Ecc has also been found to produce polygalacturonases. Nasuno and Starr (208) purified an endoPG from the culture fluid of Ecc and was able to separate it from PL activity. The enzyme had an optimum pH between 5.2 and 5.4. It had an endo mode of action and produced primarily mono and digalacturonic acid. The enzyme was not stimulated by CaCL, MgCL, or BaCL and was specific for non-esterified polygalacturonic acid.
Stack et al. (271) purified this enzyme by isoelectric focusing, but they were unable to separate it from PL. However, they could assay for it under conditions that gave no PL activity. They also found it to be an endo enzyme, producing saturated dimers to hexamers. Zink and Chateijee (311) report having cloned the gene for this enzyme.
Ried and Collmer (250) used substrate overlays to detect the extracellular production of this enzyme by the three Ecc strains examined. They found a single band of activity in an extremely alkaline region, indicating the enzyme had a pi of about 10.2. They also found the enzyme to be an endo enzyme.
There are also reports of an intracellular exoPG from Erwinia aroideae. This organism has since been reclassified and is now considered to be Ecc. The enzyme attacks the non-reducing end of oligomers to produce saturated digalacturonic acid
(120). It prefers saturated oligomers to unsaturated oligomers and has a pH optimum of 7.2 (121). It can act on unsaturated oligomers to release unsaturated dimers.
Patterns of Pectic Enzyme Production. There have been many studies examining the distribution patterns of pectic enzyme production in Ecc strains grown on galacturonan. Some of these studies have produced conflicting results, although some of these differences may reflect growth differences of the culture at the time of
sampling (49). 27
Kegoya et al. (148) found that the intracellular PL activity of strain P-2 increased sharply with increasing growth, but they observed a long lag before the beginning of
PL secretion. Chateijee et al (39) observed that during the early log phase of EC 153, very little of the PL synthesized was released into the medium. During log phase,
Moran and Starr (199) found that less than 25% of the total PL of this strain was present in the culture fluid. However, after 12 hours of growth on galacturonan, 72% of the PL produced by this strain was found in the medium (39). Of the enzyme that remained intracellular, 5-10% was releasable by osmotic shock; the rest remained cell-bound.
The PL production by EC 153 is typical of most of the strains studied. When
EC201 was grown to late log phase, 83% of the PL activity was in the medium. None of the intracellular activity was in the periplasm (97). Less than 10% of the total PL produced by strain 8061 remained in the cells during log growth. Atypical results were obtained by Kegoya et al. (148) who found that only 2-3% of the PL synthesized was secreted into the culture fluid.
Very little work has been done on the distribution of PG in Ecc. Kegoya et al. found that PG is secreted during early log phase and accumulates in a fairly large
amount in the culture fluid before the beginning of PL secretion. However, they
incorrectly identified the PG as an exo enzyme. ExoPG is produced intracellulaiiy
(120), but the extracellular PG is an endo enzyme (208, 271).
Pectic Enzyme Production by Erwinia carotovora subsp. atroseptica
Very little work has been done on the pectic enzymes produced by Eca.
Rombouts (258) demonstrated that the organism produces extracellular endoPG and
endoPL. Hall and Wood (112) compared the PL from the culture filtrate of Eca with the 28
cell-free extracts from potatoes rotted by Eca. The crude PL activity was greatest at pH 9-10, and 1 mM CaCl^ was required for optimal activity. The conditions for the rot extract to cause cell separation and death corresponded to the conditions optimal for pectolytic activity.
Quantick et al. (239) isolated CWDE from culture filtrates and extracts of potatoes rotted by Eca and purified the enzymes by isoelectric focusing. They obtained three enzymes from the culture filtrate with pi’s of 9.7, 10.2, and 10.35. All of the enzymes had a pH optimum of 9.0. Only one enzyme was obtained from the rot extract. It had a pi of 10.4 and was less inhibited by chlorogenic acid than the other enzymes. Ried and Collmer (250) used substrate overlays to examine the extracellular pectic
enzymes produced by Eca. They examined three strains and found that all of them
produced 3 PL enzymes. These enzymes appeared almost identical to those produced
by Ecc, with pi’s of approximately 9.0, 10.0, and 10.3 (251). These strains also
produced an endoPG with a pi of approximately 10.2.
Weber and Wagoner (306) found that PG was the initial enzyme obtained from
potato tubers infected with Eca\ PL was only produced when the pH shifted to the
alkaline range. Large amounts of maceration were produced before PL activity could
be detected, suggesting that PG may be responsible for the initial maceration. The
pH optimum of this enzyme is 5.0.
Pectic Enzyme Regulation
Many organisms produce pectic enzymes but are not plant pathogens. The
pectolytic plant pathogenic bacteria are better able to rapidly synthesize and secrete
large amounts of pectate lyase in the presence of host tissue or pectic compounds (39,
313). The patterns of pectate lyase regulation in the soft rot Erwinia may be an 29
important factor in determining pathogenicity.
The soft rot Erwinia have evolved a complex regulatory system for pectic enzyme synthesis and secretion. The bacteria constitutively produce a low level of PL, but they are induced to produce and secrete large amounts of PL in the presence of
uronic acid compounds. An excess of the breakdown products of the enzymes causes
self-catabolite repression and limits PL production.
Catabolite Repression. The term "catabolite repression" describes the
phenomenon in which "the presence of glucose or other rapidly metaboUzable
substrates in the growth medium elicits a more or less severe but permanent
repression of catabolic enzymes" (292). In E. coli, catabolite repression has been
linked to the levels of intracellular cyclic AMP (cAMP) in the bacteria. The levels of
cAMP decrease during catabolite repression and increase after its removal.
The increased concentration of cAMP facilitates the binding of this compound to
a protein known as the catabolite activator protein (CAP). This complex interacts
with the promoters of "cataboHte-sensitive" promoters and stimulates transcription of
the corresponding operon. Mutations in the adenylate cyclase gene (cya) or in the
gene coding for CAP (crp) result in bacteria which are unable to use a variety of
carbohydrates as carbon sources. The addition of exogenous cAMP restores growth on
all carbon sources in cya mutants, but not in crp mutants. The cAMP-CAP complex
has also been implicated in rho-dependent termination of transcription.
PL production in Ecc and Ech has been shown to be subject to catabolite
repression by glucose (39,199, 313), and the addition of glucose to Ecc cultures
growing on galacturonate caused a decline of PL production. This decline correlated
with a decline in intracellular cAMP concentration and could be reversed with
exogenous cAMP (131). Mount et al (206) isolated a cAMP-deficient mutant of Ecc 30
EC 14 that was unable to produce PL during incubation with sodium polypectate unless exogenous cAMP was supplied. This mutant was presumably a cya mutant, since the organism was able to respond to the addition of cAMP. These bacteria can also undergo self-catabolite repression when PL generates products more quickly than the cell can utilize them (46).
The mechanism of catabolite repression in Ecc and Ech appears to be similar to that of £. coli. The genes of pelB and pelE of Ech have sequences that would be expected to function as CAP-binding sites in E. coli, and the deletion of these sites from the cloned genes abolished catabolite repression in E. coli (145). The cya gene from Ech complemented adenyl cyclase-deficient E. coli mutants (123). pelE and a putative pelC gene contain sequences at the 3’ end that resemble rho-independent transcription terminators (147), and Ecc and Ech have been shown to contain rho termination factors similar to those of E. coli (27, 213).
Induction. The polysaccharides in native cell walls are insoluble, and, as such, cannot induce the production of the enzymes which degrade them. However, the constitutive production of a low level of PL enables the soft rot bacteria to partially degrade the pectin from cell walls and allows induction to take place.
PL induction can be induced by growing the bacteria on isolated plant cell walls
(47), PGA, gluconate, galacturonate, or glycerol (39). There is a lag after the addition of polygalacturonate to cultures before PL induction begins (289), suggesting that pectic acid itself does not induce PL synthesis. The inducer appears to be a breakdown product of polygalacturonate. Tsuyumu et al (290) found that the addition of unsaturated dimer to cultures of Ecc caused rapid induction and that the addition of large amounts of the dimer resulted in catabolite repression. Large amounts of digalacturonate do no have this effect (40, 47). 31
The addition of EDTA to Ecc cultures prolongs the lag before PL synthesis is stimulated, suggesting that PL activity is responsible for induction (289). However,
EDTA does not inhibit this induction when cells of Ech are incubated with soluble D- galactmonate (41). The exoPG produced by Ech appears to be involved in this induction; it can generate unsaturated dimers from unsaturated oligomers. However, when the cells were incubated with isolated plant cell walls, EDTA did inhibit PL induction.
Chateijee et al. (40) suggested that a common metabolite derived from either saturated or unsaturated dimer induced PL production, and Collmer and Bateman (47) studied the induction of PL in an Ech mutant deficient in OGL production. This mutant was poorly induced unless it was supplied with DKH or unsaturated trimer that could be converted to DKI by intracellular exoPL. They proposed that induction on
D-galacturonan required the release of dimers from the polymer as an intermediate step in deoxyketuronic acid formation and that the deoxyketuronic acids are the apparent inducers of PL.
It is not clear which of the metabolites of the deoxyketuronic acid pathway is responsible for the induction of PL. Mutants deficient in OGL production were not induced to produce PL by isolated plant cell walls, polygalacturonate, or saturated or unsaturated dimers, but they were induced by exogenous DKH (41,47). PL was only weakly induced by monomeric galacturonate, suggesting that KDG was not very effective as an inducer (47). This would suggest that DKI or DKH, or both, are the actual inducers. Evidence for this was provided by studies of kduD" mutants in Ech that were unable to convert DKII to KDG and were hyperinduced by PL in the presence of dimers (41, 54). However, Condemine et al. (54) have shown that KDG can also function as an inducer of PL. Further evidence for this was provided by 32
Hugovieux-Cotte-Pattat and Robert-Baudouy (245) who mutagenized each gene involved in hexuronate catabolism. The various intermediates were not able to induce PL synthesis by themselves, but after transformation to KDG, they could act as inducers.
Also, when KDG accumulated in a kdgK mutant strain, PL synthesis was highly induced.
The galacturonate pathway appears to be regulated independently of the deoxyketuronic acid pathway (54). The genes involved in galacturonate catabolism in
Ech have been cloned into E. colU and Ech mutants deficient in these genes have been isolated (41,134, 297). Saturated dimers induce PL production in these mutants.
In addition to the genes to metabolize galacturonate {uxaC)y tagaturonate {uxaB)y and altronate (uxoA), there is also a gene, exuTy whose product specifically transports galacturonate and glucuronate into cells. All of these genes are grouped in an operon in Ech 3937 and are under the control of a regulatory gene product {exuR), which probably acts as a negative regulator (134). This product did not affect PL synthesis.
The regulation of the genes involved in KDG breakdown was also studied (134).
The genes for the transport of KDG into the cell (kdgT) and the metabolism of KDG
(kdgK) and KDGP (kdgA) were found to be controlled by the product of kdgR. This product affected other genes of PGA catabolism, such as kduDy ogh and the pel genes, but it did not affect the expression of galacturonate or glucuronate catabolism. The
PL genes were also affected by other regulatory factors, since the synthesis of PL remained inducible after the inactivation of kdgR. The transport system for KDG can also mediate the entry of DKI and DKII; they had a higher affinity for the transport system (55). KDG is not transported into wild-type cells, and it is suggested that the transport system be called the ketodeoxyuronate transport system. In mutants lacking this transport system, DKI and DKII did not induce PL synthesis. These mutants grew 33
more slowly on polygalacturonate, and less PL was induced. JcdgT is subject to catabolite repression by glucose; this can be lifted by the addition of cyclic AMP (55).
Proposed Model for Pectate Lyase Regulation in Ech. A review of the literature suggests that there is differential regulation of some of the pectate lyase enzymes of Ech. I would like to suggest that when the cells are relieved from catabolite repression, they preferentially synthesize PLb and PLc. These enzymes are secreted, and if they encounter pectic compounds, they release unsaturated dimers.
These dimers then induce the production of PLa, PLd, and PLe.
The role of PLb and PLc in soft rot pathogenesis is not clear. Although PLb and
PLc act in an endo manner, they also produce a lot of exo activity and do not macerate tissue as efficiently as the other pectate lyases (99). Mutants which lacked
PLb or PLc were able to macerate potato tissue as well as the parent strain (257)
Also, a different strain which lacked PLb was as pathogenic as the wild-type on
Saintpaulia (28).
However, these enzymes are consistently produced, suggesting that they are useful to the organism. Cooper (58) has suggested that endo enzymes which act in a dual fashion by initially breaking a chain at random, then progressing along the polymer releasing low MW polymers would be efficient at generating products for induction.
The mode of activity of these enzymes seems analogous to that described by Cooper
and suggests that they may serve to induce more PL production. Some evidence for
this is provided by mutants lacking PLc which took one week longer than the wild-
type to produce symptoms on Saintpaulia (28).
The genes for PLb and PLc both have strong promoters: a clone of pelB was
shown to have a very strong promoter in E. coliy although it lacked the palindromic
sequence of pelE that would be expected to function in induction (146, 147). This 34
may indicate that the pelB gene would be more likely than the pelE gene to be transcribed when catabolite repression is lifted. The pelC gene from several strains appears to be expressed better in E. coli than other genes when controlled by its own promoter (50). pelB and pelC have a lot of homology and are very close to each other; they are separated from pelA, pelD, and pelE^ which are also close together
(155). Since pelB and pelC are next to each other, this may indicate that they are coregulated, and their separation from the other PL genes suggests that they may be regulated differently.
pelE has no identifiable strong promoter (147), although it has a palindromic
sequence at the 5’ end that may be involved in induction (146). This suggests that
the gene is not automatically transcribed when catabolite repression is lifted, and that
it requires induction to be transcribed. Also, the signal sequence has a longer N
region and is more basic than that of pelB, which may indicate a different mode of
secretion (147). The genes from PLa, PLd, and PLe are all grouped together, which
may indicate that they are regulated in similar manner. If this is so, they should all
be induced together by products generated from PLb, PLc, or exoPG.
PLd and PLe attack pectic polymers in a more random manner and seem more
destructive to plant tissues than the neutral enzymes (50). E. coli clones of the pelE
gene can macerate as effectively as Ech (147) and can multiply in inoculated potato
tubers (215). These enzymes are the primary ones associated with the maceration characteristic of soft rot disease, and, teleologically, it would seem reasonable for
them to be induced by the action of previous enzymes.
Hugovieux-Cotte-Pattat et aL (133) have isolated two classes of regulatory mutations that affect the synthesis of PL in Ech, One class (cri) are not affected by catabolite repression: PLb and PLc synthesis became resistant to catabolite 35
repression. A low level of PLa, PLd, and PLe were also produced under conditions of catabolite repression (245). This mutation was not specific for pectic enzymes; the expression of other genes was affected, including those affecting cellulase and protease production. The other class of mutations {gpi) were no longer inducible in the presence of pectin derivatives: PLa, PLd, and PLe were mainly affected. This suggests that the basics of this model are correct.
However, recent research has indicated that the regulation of pelA, pelDy and pelE are very complex (245). Each is controlled at different levels, by each of the
kdgR, gpiR, and cri gene products. Each of these genes is regulated differently,
although they may still all need to be induced by metabolites before they are
expressed. pelD is induced more strongly by KDG than the other genes, and in cri
mutants, PGA induces a higher level of production than in the wild-type strain. The
cri gene may not be directly responsible for this induction; the release of catabolite
repression may have resulted in an increased release of real inducer from PGA.
Unlike pelE^ pelD is not fully expressed in kdgR or gpiR mutants, indicating that they
have only partial control over the production of PLd. pelA expression is only slightly
affected by kdgR and gpiR mutations, and they do not seem to be the essential
regulatory genes governing PLa synthesis. Also, a mutant lacking kdgK selectively
overproduced PLb and PLc when incubated with polygalacturonate (296), and the ratio
of PLd to PLe was lower in non-induced cultures than when the enzymes were
induced by PGA. 36
Proteases
Plant Cell Wall Protein
The primary cell walls of dicots are composed of 5-10% protein, which is exceptionally rich in hydroxyproline (62, 73). Most of these proteins are glycoproteins, and make up a heterogeneous group referred to as hydroxyproline-rich glycoproteins (HRGPs). There are two main groups of these proteins—structural proteins such as extensin, and arabinogalactan proteins. The protein content of secondary walls is negligible (161).
Primary cell walls contain an insoluble glycoprotein known as extensin: a highly basic protein with an unusual amino acid composition (187). It is rich in hydroxyproline and also has a relatively high content of alanine, serine, and threonine
(60, 73). Two-thirds of the molecule is carbohydrate, and, in dicots, almost all of the hydroxyproline is glycosylated with arabinose, primarily tetra-arabinoside (73). This
sugar comprises 96% of the carbohydrate of the molecule. The terminal arabinose is a-linked, while the rest are 6-linked. The rest of the carbohydrate is made up of
galactose, which is attached singly by an a-linkage to some of the serine OH groups.
Extensin does not contain arabinogalactan.
The structure of this molecule is that of a polyproline-II helix, (a left-handed
helix with three residues per turn) which is stabilized by its carbohydrate side chains
(56, 129, 300). Extensin is an 80 nm long rigid rod and contains a characteristic repeating pentapeptide sequence, Ser-(Hyp)^ (77,159, 300). The compound is highly
insoluble, and remains in the shape of the original cell wall after treatment by
anhydrous hydrogen fluoride, which depolymerizes the carbohydrate of the plant ceU wall (201). However, extensin could be solubilized by treatment with acidified
NaClO^, a compound that cleaves phenolic oligomers but not peptide bonds (161). 37
Extensin has recently been shown to contain isodityrosine (93), and it is thought that
the tyrosyl residues of adjacent molecules are linked by diphenyl ether bridges (96).
There is strong evidence that this reaction occurs and is catalyzed by wall-bound peroxidase, but the existence of intermolecular isodityrosine bridges has yet to be
definitively proven (96).
The function of extensin is plants has not been fully determined. There is
evidence that the level of extensin is developmentally regulated (302), and it has been
thought to restrict cell expansion, because of its tight cross-linking. Extensin
content has been negatively correlated with growth rate (187). The insoluble cell wall
glycoprotein was originally proposed to be linked to the pectic rhamnogalacturonan
fraction through arabinogalactan (142), but this has since been found not to be the
case. The involvement of the HRGP was inferred from the structure of secreted
HRGP, which has since been found to be different from the insoluble HRGP (22).
Currently, there is no good evidence that the cell wall HRGP is covalently bound to
other cell wall constituents.
However, it is thought that extensin interacts noncovalently with cell wall
polysaccharides, although the mechanism is not known (60, 73, 187). Lamport and
Epstein (162) have proposed a model of primary cell wall structure in which cellulose
microfibrils coated with xyloglucan penetrate the pores of a network of extensin
molecules connected by isodityrosyl bridges; there appears to be a strong association
between protein and cellulose microfibrils (81,195). In addition, the basic nature of
extensin indicates that it could interact with the acidic pectins (162). Molecular
modeling studies of the extensin precursor from carrot have shown a repeated set of
positive charges which are very similar to the spacing of the two acidic groups of
polygalacturonic acid (56). An extensin-pectin network may be part of the primary 38
cell wall structure in dicots.
Extensin is highly resistant to proteases, and may help to protect the cell wall against degradation. Even after severe enzymic attack by a mixture of hemicellulase, cellulase, and protease, much of the extensin remained as an insoluble wall-shaped residue (157). It could be cleaved by trypsin only if the arabinosyl residues were first removed by acid hydrolysis (162), and the B-linked arabinoses are thought to hydrogen bond with the hydroxyproline residues to form a "nest" that shelters the peptide linkages (160). Virtually all of the arabinosidases that have been isolated are specific for a linkages (161), and the arabinosyl residues probably help to protect the polypeptide from enzymic degradation. In addition, few enzymes can cleave the Hyp-
Hyp linkage (161).
Cell wall HRGPs often increase in response to pathogens and have been correlated with resistance to some. The enhanced resistance of potato and tomato to nematode attack corresponded to increases in the amounts of wall-bound hydroxyproline (103,
310). In melon seedlings infected with Colletotrichum lagenariwn (83) artificial enhancement or suppression of the cell wall HRGPs respectively increased or decreased resistance to the fungus (83). Glycosylation of the hydroxyproline residues increased following infection, and the proteases secreted in culture by the fungus were ineffective in extracting HRPG without prior deglycosylation of the hydroxyproline residues (81, 82).
The enhancement of HRGPs in cucumber cell walls has been considered as an
early event in the resistance of cucumbers to Cladosporium cucumerinum (114),
Resistant cultivars were found to have earlier and greater increases in cell wall HRGP
than susceptible cultivars (114). Resistance of cucumbers to this fungus had
previously been shown to be strongly associated with the rapid lignification of the 39
host’s cell walls (113), and it was suggested that these additional HRGPs may act as matrices for lignification (307).
Some plants contain soluble HRPGs that are ionically bound to the cell wall and are capable of agglutinating bacterial cells (160). Many unrelated gram-negative bacteria have been found to bind to tobacco cell walls (265). Certain avirulent strains of Pseudomonas solanacearum are rapidly attached to and enveloped by the cell walls of tobacco and potato, while virulent cells remain free in the intracellular spaces and multiply rapidly (265). Virulent strains remain free or are agglutinated only weakly
(266), and a very strong correlation has been shown between virulence and the presence of an extracellular polysaccharide slime (EPS) in this bacterium (135). A similar correlation has been found for Erwinia amylovora and E. stewartii (13). Some suggest that EPS may prevent binding and attachment of the bacteria to the cell walls
(253a), but others disagree (237). There has been some speculation that the immobilization of bacteria may result in a hypersensitive response, however immobilization does not appear to be a universal response (191, 237). The exact role of agglutinins in the induction of resistance to bacteria is not clear (156).
The agglutinins that have been isolated from potato tissues (167) and tobacco callus (188) have amino acid and carbohydrate compositions similar to that of the cell wall HRGP in carrot, and it seems likely that they have a similar conformation (301).
There is some evidence that these soluble HRGPs are precursors to extensin (57), and the HRGP from carrot was found to have the same agglutination titer as the agglutinin from potato (300). The proteins are thought to bind to the lipopolysaccharide (EPS) of the bacteria (266), and agglutination appears to be by a charge-charge interaction rather than by the sugar-specific interaction characteristic of lectins (166). The highly basic nature of the protein allows it to function as a 40
polycation.
Touz6 and Esquerr6-Tugay6 (266) have suggested that HRGPs may play a role in the general defenses of plants against pathogen attack. HRGP was found to increase in response to infection of a variety of hosts by fungi, bacteria, and viruses in all of the dicots studied, but the extent varied according to the host species (181). This increase must occur early during the infection process to be effective, and it has been pointed out that the accumulation of HRGP has some similarities to a phytoalexin response (181).
HRGP accumulation is increased in race-cultivar specific resistance (114), and synthesis can be elicited by elicitors of both fungal and plant origin, including some which also elicit known phytoalexins (256). In incompatible interactions of C. lindemuthianwn with Phaseolus vulgaris, HRGP mRNAs increased markedly during the early stages of infection and correlated with the expression of hypersensitive resistance (267). In the compatible reactions, HRGP mRNA increased later during the infection process, at the onset of lesion formation. The HRGP also accumulated in healthy tissue distant from the initial site of inoculation, implying the transmission of an elicitation signal. While increases in cell wall HRGP have been correlated with resistance in some systems, levels were increased more in wheat by compatible interactions of Erysiphe graminis (44). Incompatible interactions did not result in these increases. It is possible that the interaction of a plant with an obligate parasite may be different than with non-obligate ones. Increased levels of HRGP are also induced by stresses such as wounding (278) and heat shock (276), and ethylene leads to greatly enhanced levels (248, 285). The exact role of HRGPs in the resistance of plants to pathogens needs to be clarified.
In addition to the extensin and the similar soluble HRPGs, the cell walls of plants 41
contain a very different group of HRGPs. These are the arabinogalactan proteins
(AGPs), a heterogeneous group of acidic proteins that are virtually ubiquitous in higher plants (160). Like extensin, they are rich in hydroxyproline and serine, and a portion of the molecule is in a polyproline 11 configuration (299). However, their amino acid composition is very different, and they contain over 90% carbohydrate, primarily large, highly branched arabinogalactan chains linked through galactose to the hydroxyproline (231). In addition, many other sugars may also be present (85).
Most of the AGPs behave as 6-lectins and bind to certain phenolic 6-glycosides
(85, 161). They differ from classical lectins in having only a single carbohydrate binding site. Not much is known about the biological role of these glycoproteins (85).
Suspension cultures secrete large amounts of them into the growth medium (231), and they may be a component of the middle lamella (161). It is interesting that a high
MW protein was released from the middle lamella of potatoes by degradation with pectic enzymes (137). This protein did not contain hydroxyproline, and Ishii (138) suggested that it joined with homogalacturonan to form the middle lamella. It was not further characterized, but it may have been an AGP. Other AGPs may be involved in pollination and fertilization (71, 161). Some are also found intracellularly and on the surface of the plasma membrane and it is suggested that they are involved in cell recognition (85, 161).
The cell walls of some plants contain true lectins: proteins with multiple binding sites that selectively bind carbohydrates without any obvious enzymatic activity (160).
Classical lectins agglutinate red blood cells. Such a lectin has been isolated from mung bean seedlings; it binds specifically to galactosyl residues (142). It has been suggested that this lectin may function in establishing a "noncovalent protein-glycan network" (62). Other lectins on the surface of pea, soybean, and clover roots have 42
been implicated as binding sites for Rhizobium {Til), Some lectins in the cell wall may function as wall-bound glycosidases (73).
Potato plants contain a lectin that specifically binds N-acetylglucosamine. This lectin is distinct from the bacterial agglutinin and binds red blood cells (167). This glycoprotein resembles extensin in being rich in hydroxyproline, galactose, and arabinose, and it may also be in the polyproline II conformation. However, it differs from extensin since it contains cysteine, and it may be located in the cell membrane
(7). Other Solanaceous plants also contain similar lectins, and immunological results suggest they are related.
Plant cell walls also contain wall-bound enzymes which serve to regulate plant growth and transport processes or to defend the plant against invading pathogens (73,
152). Chitinase and 6-glucanase have been isolated from many plants and have been implicated in disease resistance (29). Plants also contain proteins that inactivate the degradative enzymes of pathogens. Some of these are in vacuoles, but others are in the cell wall (29).
The cell walls of various dicots have been shown to contain ionically bound proteins that completely inhibit fungal endoPGs (3). The original experiments indicated that the host inhibitors selectively inhibited the PG of its pathogens, but not non-pathogens (3, 86). However, now it is thought that the inhibitors are relatively non-specific to PGs from different races and species of pathogens, and their role in resistance is not clear (59). It is possible that these inhibitors are present to limit plant PG activity. Fungal PGs can ionically bind to some plant cell walls, and it has been suggested that proteins are involved in this (59, 286). This binding may increase the fungus’s pathogenicity by restricting the degradation of the pectin to the vicinity of the pathogen, thus limiting the elicitation of defense reactions (286). 43
These discussions have involved the cell wall proteins from dicots; these proteins are very different from those in monocots (62). Although the amount of protein in the walls of suspension cell cultures of monocots is equal to or greater than that in dicots, the walls of monocots contain much less hydroxyproline. Of the hydroxyproline that is found, most is not glycosylated. When side-chains of arabinose are present, they are predominantly tri-arabinosides (62). In contrast, dicots contain primarily tetra-arabinosides.
Classes of Proteases
Proteases are enzymes that hydrolyze the peptide bonds in protein. The substrate specificity of these enzymes is extremely difficult to define (17), so they are classified into four groups based on the catalytic mechanism of their active site (180).
Serine proteases contain serine at the active site and are inhibited by diisopropylphosphofluoridate (DFP) and by phenylmethylsulfonyfiuoride (PMSF); they seem to be the most numerous group (17). Proteases with cysteine at their active site are known as thiol proteases and are inactivated by thiol reagents; they are rarely produced by microorganisms (22). Metal chelator-sensitive proteases require bound metal ions for activity and are inactivated by chelating agents.
The last group is traditionally referred to as acid proteases and are characterized by a low pH optimum. This group consists primarily of enzymes with aspartic acid and tyrosine at their active site, and are known as aspartic acid proteases (17). They seem to be found only in eukaryotes (17). Storey (277) advises against using the term
’’acid protease", since it does not distinguish between aspartic acid proteases and thiol proteases with an acid pH optimum.
Involvement in Plant Pathogenesis
The production of proteases by plant pathogens has been known for years, and 44
protease activity is often found in diseased tissue (17). In some cases, this activity was shown to be derived from the pathogens (127, 230, 293), and the important structural and defensive roles of proteins in plants suggests that proteases produced by pathogens could play important roles in pathogenesis.
Proteases that degrade plant cell wall protein during the early stages of pathogenesis should facilitate maceration, but the glycosylation of HRGPs make them very resistant to degradation (157, 161). Proteases from Monilinia fructigena and
Colletotrichum lindemuthianwn were unable to liberate HRGP from the cell walls of their hosts (127, 252), while protease from Erwinia carotovora was unable to macerate potato or cucumber tissue (205).
Another possible role of proteases in pathogenesis could be to degrade the host’s bacterial agglutinins or the inhibitors of pathogen CWDE. The agglutinins have structures similar to extensin (301) and would also be difficult to degrade.
Colletotrichum lindemuthianwn has been reported to produce a protein that inhibited the PG inhibitor produced by kidney bean, but there is no mention of the protein having enzymatic activity (4).
The proteases examined so far have not been shown to be essential for pathogenicity. A mutant of Cladosporium cucunerinum that did not produce protease was as effective as the parent strain at causing disease (255). However, even if they are not necessary for pathogenicity, proteases may still contribute to the pathogenicity of an organism. A protease from E. carotovora caused the cells of cucumbers to burst, suggesting that it may have toxic effects on membranes (287).
An extracellular protease produced by Pseudomonas syringae pv. tomato may contribute to symptom development in tomato plants (20). It was suggested that the products from the enzyme activity were deaminated, producing ammonia, which is 45
thought to contribute to necrosis of the host tissue.
Even if they are not directly involved in pathogenesis, proteolytic enzymes probably add to the nutrition of the organisms, either in the invaded tissue or in soil.
The acid protease from Monilinia fructigena was shown to be present in infected apple fruit and absent from healthy fruit. It was suggested that the protease had a nutritional role in vivo, where the pH of infected, injured, and dead tissue is similar to the pH optimum demonstrated in vitro (127). A similar role has been suggested for
the protease from C. lindemuthianum (252). Protease activity from C. lagenarium in
melon seedlings increased three days after inoculation, when the pathogen was already
present in the host cells (230). This may indicate that the protease was functioning
to provide nutrients for the fungus.
Protease Inhibitors in Plants
Protease inhibitors are found in a wide variety of plants, and are often found in
high concentration in many seeds and other storage organs (259). Inhibitors of serine
proteases are especially common (261), but very few inhibit acid proteases (247).
These inhibitors may function as storage or reserve proteins, to regulate protein
turnover and metabolism, or to defend against attacks of insects or microorganisms
(247). A few endogenous inhibitors of plant proteases have been isolated, but they
are present in low amounts (260). The high concentration of serine protease
inhibitors suggests that their function may be one of plant protection.
Potato tubers are a notable for their wide diversity of proteinase inhibitors
(247), and those most thoroughly studied for their role in defense are inhibitor I and
inhibitor 11 from potatoes and tomatoes. These are potent inhibitors of serine
proteases and metallo-carboxypeptidases and do not inhibit any known plant proteases
(260). They are developmentally regulated in potato tubers and stored there in high 46
amounts (261). They are synthesized by both potato and tomato leaves in response to
any type of severe wounding. A signal, known as the proteinase inhibitor inducing
factor (PIIF), is released at the wound site and transported to other leaves. It
initiates the synthesis and accumulation of the two inhibitors, which accumulate in
both wounded and unwounded leaves throughout the plants (261). PIIF was found to
be a small pectic polysaccharide with an amino acid composition similar to that of
rhamnogalacturonan H (262). PIIF active oligosaccharides can be released from
isolated tomato leaf cell walls by an endoPG partially purified from tomato plants (26).
Some of the plant protease inhibitors inhibit the proteases of plant pathogens,
suggesting that they may protect plants from proteases. Mosolov (202) demonstrated
inhibition of extracellular proteases of Fusarium solani by protease inhibitors from
many plants. Peng and Black (217) showed a correlation between an increase of
protease inhibitor activity in tomato plants following infection and resistance of those
plants to Phytopthora infestans. Infection with incompatible races resulted in a rapid
and persistent increase in protease inhibitor activity, while compatible reactions
resulted in lower activity. In another system, kidney bean seeds were shown to
contain a protease inhibitor highly specific to a protease secreted in vitro by
Colletotrichwn lindemuthianwn (203).
Production of Proteases by Erwinia
Production of proteases has been demonstrated in several species of Erwinia that
cause very different types of diseases. Erwinia amylovora, the causal agent of fire blight produces two proteases. Both proteases are present in the ooze from infected immature pear fruits, and Protease n is also produced in culture. Protease I has a pi of 9.9, a pH optimum of 7.5, and is totally inactivated by 10 mM EDTA. Protease II has a pi of 4.6, a pH optimum of 6.5, and is also inhibited by 10 mM EDTA. This 47
enzyme can be extracted from infected plant tissue. Both enzymes fit in the class of
metal chelator-sensitive neutral proteases.
Two species of the soft rot Erwinia have also been shown to produce proteases.
As mentioned, Ecc produces a protease which is unable to macerate cucumber or potato tissue, but can lyse cucumber protoplasts (205, 287). This organism produces
an extracellular protease which is eluted in the 0.4 M NaCl gradient of a DEAE column (205). Subsequently, another protease was isolated from this organism; this enzyme is eluted in the void volume of a similar column and has a pi of 8.3 (287). It is possible that the latter protease is an intracellular enzyme which had been released
by autolysis, since the bacteria were grown for 14 days before the enzyme was
extracted.
Recent studies on Ecc EC91 have shown a protease which is eluted in the 0.3-0.4
M NaCl gradient of a DEAE column and has a pi of 4.95 (268). This is presumably the same enzyme as that isolated by Mount et al. (205). Genetic studies have been conducted on this enzyme, and a cosmid containing the prt gene was used to complement an Ecc prt- mutant (8).
Erwinia chrysanthemi also produces extracellular proteases, and Garibaldi and
Bateman (100) demonstrated that the amount of activity varied greatly between strains. Those from chrysanthemum and Diejfenbachia had very high activity, while those from com and carnation had low activity.
Wandersman et al. (304) isolated a hyperproteolytic mutant of Ech that produced two proteases. One was a 50 kD protein with a pi of 5.8 and a pH optimum of 6-8.
The enzyme was a neutral serine protease which was totally inactivated by 10 mM
PMSF, although not by 10 mM EDTA. The other enzyme was a 55 kD protein with a pi of 4.6. The two enzymes were unrelated antigenically, and intracellular activity 48
was not detected. The enzymes were produced only in cultures induced with peptides.
In the wild-type strain, the 55 kD protein was produced in low amounts, but the 50 kD protein was rapidly converted to inactive but immunologically cross-reacting degradation products.
The protease of Ech appears to be secreted differently than other extracellular
enzymes produced by the organism. Mutants which could not export pectate lyase,
polygalacturonase, or cellulase were able to secrete protease (10, 283). In addition,
Escherichia coli clones which contained prt and pel genes secreted protease, while
pectate lyase remained in the periplasm (16, 283). The characteristics of the secreted
protease were not determined; it is not clear whether one or both of the proteases
were secreted. Chapter 2
CELLULAR LOCALIZATION AND CHARACTERIZATION OF PECTIC
ENZYMES
Introduction
Erwinia carotovora subsp. atroseptica (Eca), Erwinia carotovora subsp. carotovora (Ecc), and Erwinia chrysanthemi {Ech) cause soft rots and other diseases of plants throughout the world. These bacteria produce an array of extracellular enzymes that enable them to degrade plant cell wall and plant cell membrane components (22). In particular, pectic enzyme production has been correlated with the maceration and cell death characteristic of soft rot disease (43, 49, 50).
The pectic enzymes of Ech and Ecc have been well studied. Both organisms produce multiple isozymes of pectate lyase (PL), and each also produces polygalacturonase (PG) (50). Purified endoPL has been shown to macerate tissue and kill cells (18, 205, 275), but the roles of individual enzymes in pathogenesis are not clear. Many of the PL genes (pel) from Ech and Ecc have been cloned (31, 147, 155,
170, 244, 253, 254, 311), as has the PG gene (peh) from Ecc (311). The development of site-directed mutagenesis techniques in Ech has facilitated studies on the roles of the individual PL enzymes in pathogenesis (28, 257).
In contrast, little work has been done on the pectic enzymes produced by Eca,
This organism is limited almost entirely to potatoes and differs from Ech and Ecc, which have broad host ranges (227). Also, Eca produces much less inducible PL per cell than Ecc (314). Both extracellular endoPL and endoPG are produced (258). By using sucrose column isoelectric focusing (lEF), Quantick et al (239) isolated three extracellular endoPL enzymes that had slightly different modes of action in degrading
49 50
pectic substances. Ried and Collmer (250) analyzed the pectic enzyme profiles of three strains of Eca: all produced three bands of PL activity and one band of PG activity in overlays of polyacrylamide (PAGE) gels. The gene for one of the endoPL’s has been cloned and shown to have strong homology with an Ecc endoPL (8). Weber and Wagoner (306) found that, after induction, extracellular PG was produced more quickly than extracellular PL, and they suggested that the degradation of pectin was started by PG.
The extracellular pectic enzymes produced by Eca have not been well- characterized, and the intracellular pectic enzymes have not been studied at all.
These enzymes are necessary for catabolism of the pectic fragments and induction of the extracellular enzymes; thus, they are important for soft rot pathogenesis. The cellular location of these enzymes may provide some clues to their roles in pathogenesis. Gardner and Kado (97) have isolated an endoPL from the periplasm of
Erwinia rubrifaciens, but the cellular location of specific Ech, Ecc^ or Eca intracellular pectic enzymes has not yet been determined. The cytoplasmic, periplasmic, and extracellular pectic enzymes produced by Eca strain SR-8 were isolated and characterized. A preliminary report of this research has been published
(101). 51
Methods
Cultural Conditions
Erwinia carotovora subsp. atroseptica strain SR-8 was obtained from A. Kelman,
Univ. of Wisconsin (226). Media for enzyme extraction contained 0.5% sodium polypectate (NaPP) and minimal salts (NaPPMS) (271). Bacteria were grown at 30“C overnight on a shaking water bath (90 rpm). A one ml sample was then transferred to each of five flasks of 200 ml of NaPPMS and grown until early stationary phase was reached. The bacteria were maintained on NaPPMS agar.
Enzyme Extraction and Purification
Cells were collected by centrifugation (20,000 g at 4*C for 20 min) using a GSA rotor in a Sorvall RC2-B refrigerated centrifuge. The supernatant fluid was saved for isolation of extracellular enzyme. The bacteria were resuspended in an equal volume of 0.05 M Tris-HCl buffer (pH 8.0) and washed by recentrifuging twice at 15’C.
The periplasmic fraction was isolated using a modification of the lysozyme-EDTA treatment (308). The cell pellet was resuspended in 0.2 M Tris-HCl (pH 8.0) (10 ml/2 g cells) and diluted with 2 V of the same buffer. 1.5 V of 1.5 M sucrose (pH 8.0) was added, followed by the gradual addition of 1.5 mM EDTA (3.3 ml/10 ml). Egg- white lysozyme (1 mg/ml) was added to a final cone, of 60 |ig/ml. The solution was diluted 2 fold with water and stirred over ice for 30 min. 1 M MgCl^ was added to a final cone, of 0.02 M to stabilize the spheroplasts, which were centrifuged as above at
4‘C. The supernatant was saved for isolation of periplasmic enzyme.
The pelleted cells were resuspended in 0.05 M Tris-HCl (pH 8.0), and lysed by ultrasonification with a Branson Sonifier cell disrupter medium tip at 30 W for 10 sec.
The cell sonicate was centrifuged as above, and the supernatant was saved for isolation of cytoplasmic enzyme. 52
To ensure that the fractions were adequately separated, each was assayed for cyclic phosphodiesterase (211), which is found in the periplasm, and B-galactosidase
(75), which is contained in the cytoplasm. The assay for cyclic phosphodiesterase involved incubating 60 pi of 0.025 M CoCl^, 60 pi of 0.5 M Tris-Mal (pH 6.7), 60 pi calcium bis-p-nitrophenyl phosphate (5 mg/ml), and 120 pi of enzyme at 37“C for 20 min. The reactions were stopped with 3 ml of 0.1 N NaOH, and activity was determined by measuring the absorbance at 410 nm.
To determine B-galactosidase activity, 4.5 ml of 0.05 M phosphate buffer (pH 7.5) and 0.2 ml of 0.032 M reduced glutathione were brought to 30‘’C. 200 pi of enzyme was added, followed by 5(X) pi of 0.01 M o-nitrophenyl-B-D-galactopyranoside
(ONPG), which had also been brought to 30*C. The reaction mixtures were incubated for 15 min, and then 1 ml of Na^CO^ was added to stop the reaction. The tubes were shaken, and their absorbance at 420 nm was determined.
The crude preparation of each fraction was brought to 95% saturation with
(NH^)2S0^ and centrifuged as above for 30 min. The precipitate was resuspended in distilled water (dH O) and dialyzed overnight at 4“C against several 1 of dH O. The dialysate (0.5 mg extracellular, 1 mg periplasmic, or 15 mg cytoplasmic) was applied to a 1.6 X 16 cm diethylaminoethyl (DEAE 53) cellulose column as described in Stack et al. (271). 3 ml fractions were collected and assayed for pectic enzyme activity.
Fractions from each peak showing activity were pooled and subjected to isoelectric focusing in an LKB 8101 Ampholine electrofocusing apparatus (LKB
Producter A B, Bromma, Sweden) and an LKB power source. The column contained
2.5% ampholine carriers of pH 9-11 and pH 3.5-10 in a ratio of 8:1 (280).
Electrofocusing was carried out at 5’C at 15 W with a 25(X) V maximum until the current had stabilized, usually in 40-48 hr. 1.25 ml fractions were collected in the 53
regions of PL activity; 5 ml fractions were collected in the rest of the column. All were treated as described (271).
Protein concentrations were determined by the Bradford protein assay, using bovine 6-globulin as a standard.
Enzyme Assays
Lyase activity was determined by the periodate-thiobarbituric acid assay (235) using a substrate of 0.6% NaPP in 0.05 M Tris-HCl buffer (pH 8.5) containing 0.5 mM
EDTA and 1 mM Ca"^ (CaCl^). 100 (xl of enzyme was incubated with 100 |il of substrate at SO'C for 1 hr or varying time, then 300 |il of 0.025 N NalO^ in 0.125 N
RSO, was added. After 20 min, 500 ul of 2% sodium arsenite was added, and the tubes were vortexed. 2 min later, 2 ml of 0.3% thiobarbituric acid (TBA) was added, and the tubes were boiled for 10 min. The absorbance at 548 nm was determined after they had cooled, and an optical density increase of 0.76 at A^^ indicated the formation of 1 mmole of D-galacturonic acid from NaPP. A unit of enzyme activity is defined as the amount of enzyme that yielded 1 mmole of D-galacturonic acid in 1 hr.
Specific activity was expressed as units of activity per milligram of protein.
Hydrolase activity was determined by the Nelson’s reducing group analysis (209) using substrate of 0.6% NaPP in 0.05 M phosphate buffer (pH 6.0) pretreated with 0.5 mM EDTA. 100 |i.l of enzyme solution was incubated with 100 |xl of substrate at 30‘C for 1 hr or varying times. 300 \i\ of water was added to the samples, followed by 300
III of solution A (Appendix 1), and the tubes were boiled for 10 min. After they had cooled, 500 |il of solution D was added, and they were vortexed again. 1.5 ml of water was added, and they were vortexed yet again. After 15-40 min, the absorbance at 500 nm was determined. 54
Characterization of Enzymes
The pH optimum of enzyme activity was determined by the TBA assay or the reducing group analysis using 0.6% NaPP in three buffer systems: 0.05 M acetate buffer (pH 4.5-5.5), 0.05 M phosphate buffer (pH 6.0-7.5), and 0.05 M Tris-HCl (pH
8.0-10.0).
To determine the ion optimal for lyase activity, 0.6% NaPP was dissolved in 0.05
M Tris-HCl (pH 8.5), and EDTA was added to a final cone of 0.05 mM to chelate endogenous ions. Ca^, Co"^, Mg"^, or Mn"^ ions (in the chloride form) were then added to a final concentration of 1.0 mM. These substrates were used with the TBA assay.
Identification of Reaction Products
Reaction products were separated on descending paper chromatograms with pyridine:ethyl acetate:wateriacetic acid (5:5:3:1, v/v) as a solvent as described by
Stack et aL (271).
Polyacrylamide Gel Isoelectric Focusing
The crude enzyme preparations were subjected to isoelectric focusing on an ultrathin polyacrylamide gel (PAGIEF) as described by Ried and Collmer (249), except that the samples were applied in the middle of the gel, and focusing was carried out at 6 W with a maximum of 2500 V. The PL enzymes were detected by a NaPP- agarose overlay which was incubated at room temp for 1 hr and then stained for 20 min with 0.05% ruthenium red. 55
Results
Enzyme Purification
The extracellular, periplasmic, and cytoplasmic fractions were each assayed for cyclic phosphodiesterase and 6-galactosidase. Typically, 90% of the cyclic phosphodiesterase was found in the periplasmic fraction, while 99.5% of the B- galactosidase was found in the cytoplasmic fraction (Table 1). This is comparable to previously published reports (10, 244, 312), and indicates that the enzymes found
extracellularly were truly extracellular enzymes and not the products of autolysis.
Chromatography of each of the fractions resulted in two peaks of PL activity
(Figure 2). Peak 1 contained the highest activity and was eluted in the void volume,
while peak 2 was eluted with 0.05 M-0.1 M NaCl. Peak 2 was small in the
extracellular and cytoplasmic fractions, but was much larger in the periplasmic
fraction. Peak 2 also contained a Nelson’s peak in each of the fractions. Peak 1 of
the periplasmic and extracellular fractions also contained a large and small Nelson’s
peak, respectively.
The peaks from each fraction were combined and subjected to isoelectric focusing
(pH 9-11). Isoelectric focusing of the extracellular fraction produced four major PL
peaks with isoelectric points of 10.2, 9.6, 9.5, and 9.4, and two smaller peaks of pi 9.2
and 8.9 (Figure 3). These enzymes will be referred to as PL 1-6 (pi 10.2-8.9). Two
Nelson’s peaks produced by polygalacturonase activity were also present at pH 10.7
and 3.9; these enzymes will be referred to as PGl (10.7) and PG2 (3.9). PGl
sometimes contained trace amounts of unstable lyase activity.
The periplasmic fractions contained large peaks of the PL’s of pi 9.5 (PL3) and
9.4 (PL4), and a PL of pi 7.1 (PL7) (Figure 4). A small amount of PG2 was also
present. The cytoplasmic fraction contained peaks of PL3, PL4, PG2, and small 56
Table 1. Distribution of enzyme activities in subcellular fractions*
Percent Total Activity
Enzvme’’ supernatant periplasm cvtoplasm
B-galactosidase 0 0.6 99.4
cyclic phosphodiesterase 2.5 90.2 7.3
*The supernatant was removed by centrifugation, and the periplasmic fraction was isolated by a modified lysozyme-EDTA treatment (308), followed by centrifugation. The spheroplasts were then lysed by sonification to release the cytoplasmic fraction.
’The fractions were assayed for 6-galactosidase activity by measuring their ability to hydrolyze ONPG (75) and for cyclic phosphodiesterase by determining their ability to hydrolyze bis-p-nitrophenyl phosphate (211) 57
Figure 2. DEAE cellulose column chromatography of extracellular, periplasmic, and cytoplasmic pectic enzymes. The (NH^)2SO^ preparation was applied to a DE53 column, and enzyme was eluted by a stepwise gradient of 0-0.2 M NaCl in 0.05 M Tris-HCl buffer, pH 8.0. Fractions were incubated with 0.6% sodium polypectate for one hr and assayed by the TBA assay at A (235) for pectate lyase activity (•) and by the Nelson’s reducing sugar assay A^ (^(39) for polygalacturonase activity (o). Absorbance Absorbance Absorbance 2.0 NaCI 10 Fraction Number 0.05 MTris-HCI 0.05 M0.10.2 PERIPLASMIC 20 30 58 59
Figure 3. Isoelectric focusing of the extracellular pectic enzymes. Fractions were incubated with 0.6% sodium polypectate for four hr and assayed by the TEA assay at (235) for pectate lyase activity (♦) and by the Nelson’s reducing sugar assay at A^^ (209) for polygalacturonase activity (<^. A^ (209)forpolygalacturonase activity(o). A (235)forpectate lyaseactivity(♦)andbytheNelson’s reducingsugarassayat incubated with0.6% sodiumpolypectateforfourhrand assayedbytheTBAassayat Figure 4.Isoelectricfocusingoftheperiplasmic pecticenzymes.Fractionswere
OvU Absorbance 0.2 0.4 0.6 0.8 1.0 Fractions (ml) 60 61
amounts of the other pectic enzymes (Figure 5). Isoelectric focusing of the entire cytoplasmic fraction resulted in PL peaks much smaller than those obtained from the purification of a portion of the periplasmic fraction. This suggests that most of the intracellular PL activity is located in the periplasm.
Characterization of Enzymes
All of the extracellular PL’s (PLl-6) produced as reaction products several, oliguronides of different sizes (Table 2), indicating an endo mode of NaPP depolymerization. The sizes of these products varied from unsaturated dimer to unsaturated pentamer, as indicated by values similar to those previously determined in this lab (271). All of the enzymes exhibited optimal activity from pH 8 through pH 10, although PLl and PL4 had greater activity at lower pH’s than the other enzymes (Figure 6).
The cation preferences of the enzymes varied. Most of the extracellular PL’s had dual preferences for Ca"^ and Co"^ ions (Table 3). PL2 was the exception; it strongly preferred Ca"^ to any other ion. All enzymes gave some activity with Mg"^ as a cofactor, but only PLl could use Mn'^. Most required ions for activity, but PL6 had lyase activity in the absence of ions.
The PL of pi 7.1 (PL7) was present only in the periplasm and produced a single reaction product, indicating an exo mode of depolymerization. The of this product was 0.70, which corresponded to an unsaturated dimer (271). The enzyme was active over a pH range of 5.5 to 10 (Figure 7) and did not require cofactors for activity, although it was stimulated by them. The enzyme strongly preferred Co"*^ ions as a cofactor, although it could also utilize Ca"^, Mg'^, or Mn'^ ions.
The polygalacturonases had similar characteristics. Each produced a series of saturated oligomers at pH 6.0, indicating an endo mode of hydrolytic depolymerization 62
Figure 5. Isoelectric focusing of the cytoplasmic pectic enzymes. Fractions were incubated with 0.6% sodium pol>j)ectate for four hr and assayed by the TEA assay at (235) for pectate lyase activity (♦) and by the Nelson’s reducing sugar assay at A^^ (209) for polygalacturonase activity (o). 63
Table 2. Chromatographic mobilities of products formed from the degradation of sodium polypectate by pectic enzymes.* purified unsaturated probable enzvme R gal, values'*- - product product formed
PG 1 0.33,0.18,0.09 - s'* trimer-pentamer
PG2 0.33, 0.18, 0.09 - s trimer-pentamer
PLl 0.70,0.43,0.018,0.08 + u dimer-pentamer
PL 2 If -1- ft
PL 3 ft + fi
PL 4 tf + tf
PL 5 If + ft
PL 6 If ft
PL 7 0.70 -1- u dimer
“Descending paper chromatography on Whatman No. 4 paper was carried out for 14-16 hr in pyridine, ethyl acetate, water, and acetic acid (5:5:3:l,v/v). Chromatograms were developed with saturated silver nitrate in acetone and 0.5 N NaOH in ethanol and washed in 6 M NH^OH (271). Reaction mixtures contained 100 |il of substrate (0.6% sodium polypectate in 0.05 M Tris-HCl buffer, pH 8.5 containing 0.5 mM EDTA, and Ca"^ to a final concentration of 1.0 mM for PL and 0.05 M phosphate buffer, pH 6.0 containing 0.5 mM EDTA) and 100 |J.l of enzyme. Incubation was at 30“C for varying amounts of time.
‘’R ^ values indicate the ratio of product migration to the distance migrated by D- gafacturonic acid standard.
TJnsaturated products in reaction mixtures were determined by the TEA assay (235).
‘^Letters indicate u=unsaturated, s=saturated. 64
Figure 6. Effect of pH on activity of endo pectate lyase enzymes. Reaction mixtures contained 100 fil of isoelectric focused enzyme plus 100 fil of 0.6% sodium polypectate in 0.05 M phosphate buffer (pH 6.0-7.5) and 0.05 M Tris-HCl buffer (pH 8.0-10.0) containing 0.5 mM EDTA and 1 mM Ca"*^. Mixtures were incubated for 14 hr (A) or for 12 hr (B). Pectate lyase activity was determined by the TBA assay (235). 65
Table 3. Effect of divalent cations on the activity of pectate lyase enzymes.
Lyase specific activity*
Purified enzvme EDTA Cq^ Mg!l Mh
PL 1 0 48.2 46.0 22.7 7.1
PL 2 0 33.8 20.1 20.4 0
PL 3 0 84.7 65.0 49.8 0
PL 4 0 53.4 49.2 24.0 0
PL 5 0 27.3 28.0 11.4 0
PL 6 12.2 55.4 54.2 46.0 0
PL 7 2.4 5.6 7.8 6.5 4.9
“Reaction mixtures contained 100 p-l of 0.6% sodium polypectate in 0.05 M Tris-HCl buffer, pH 8.5, which had been pretreated with 0.5 mM EDTA. Specific ions (Ca"^, Co'^, Mg'^, or Mn"^) were then supplemented to a final concentration of 1.0 mM. Mixtures were incubated at 30*C and analyzed by the TEA assay (235). Specific activity is expressed as units per milligram of protein. One unit is equal to one millimole of D-galacturonic acid formed per hour. 66
Figure 7. Effect of pH on activity of exo pectate lyase. Reaction mixtures contained 100 pi of isoelectric focused enzyme plus 100 pi of 0.6% sodium polypectate in 0.05 M acetate buffer (pH 5.5-6.0), 0.05 M phosphate buffer (pH 6.0-7.5), and 0.05 M Tris-HCl buffer (pH 8.0-10.0) containing 0.5 mM EDTA and 1 mM Ca"^. I^xtures were incubated for 17 hr. Pectate lyase activity was determined by the TEA assay (235). 67
(Table 2). The smallest oligomer produced was saturated trimer. Both had a similar pH range and a pH optimum of 6.5 (Figure 8). PGl contained traces of unstable lyase activity, while PG2 did not.
Polyacrylamide Gel Isoelectric Focusing
Initially, the samples were applied at the anode, as described by Ried and Collmer
(249). Overlays of the gels run in this manner revealed three main bands of PL activity, with occasional other bands. The bands were found in all three fractions.
When the samples were applied in the middle of the gel, the pattern of PL activity was different: approximately six to seven bands were detected in each fraction, all in the alkaline region (Figure 9). 68
(O <
Figure 8. Effect of pH on activity of polygalacturonase enzymes. Reaction mixtures contained 100 |il of isoelectric focused enzyme plus 100 |il of 0.6% sodium polypectate in 0.05 M acetate buffer (pH 4.5-6.0) and 0.05 M phosphate buffer (pH 6.0-7.5). Mixtures were incubated for 15 hr. Polygalacturonase activity (□, PGl; ♦, PG2) was determined by the Nelson’s reducing sugar assay (209). 69
Figure 9. Isoelectric focusing of the extracellular (A), periplasmic (B), and cytoplasmic (C) pectate lyase enzymes on a polyacrylamide gel. The enzymes were detected by placing a pectate-agarose overlay over the gel for 1 hr and staining with 0.05% ruthenium red for 20 min. 70
Discussion
The pectic enzymes produced in the cytoplasmic, periplasmic, and extracellular fractions of Eca SR-8 have been isolated and characterized. This strain produced six endoPL’s, an exoPL, and two endoPG’s. All of the endoPL’s (PLl-6) were found extracellularly, while PL3 and PL4 were also found in the cytoplasm and periplasm.
The periplasm also contained an exoPL (PL7). Both PC’s were found extracellularly, but PG2 was also found intracellularly. This enzyme was located primarily in the cytoplasm, but small amounts of it were also found in the periplasm.
NaPP overlays of PAGIEF gels revealed several different PL enzymes. Gels which were treated in the manner of Ried and Collmer (249), in which the samples applied at the anode, gave results similar to theirs. They found the extracellular fraction of three strains of Eca to produce three bands of PL activity (250). I obtained three main bands and occasional other bands from the extracellular, periplasmic, and cytoplasmic fractions. However, gels on which the samples were applied in the middle of the gel gave different results: approximately six to seven bands of PL activity were detected in each of the fractions.
This difference may have been due to the adsorption of proteins to the filter paper on which they were applied or to the inactivation of the enzymes at the low pH found near the anode (303). When the samples were applied to the middle of the gel, only one third as much protein was required to produce visible bands as when they were applied near the anode. This suggests that protein may have been adsorbed to the filter paper.
PL bands were found in each fraction, although the intensity of the bands varied.
Preparative lEF had revealed trace amounts of each endoPL in all fractions. The overlay method is very sensitive, and may have been picking up trace amounts of the 71
endoPL’s. The exoPL was not observed; it’s mode of action may have prevented if from degrading enough pectin to be detected after one hour of incubation. Also, the exoPL preferred Co"*^ ions as a cofactor, and the Ca'^ ions used in the overlays may not have activated the enzyme sufficiently.
The exact pi of the enzymes cannot be determined from these overlays, since the
gels were not run in an oxygen-free atmosphere (51). However, the overlays do corroborate the findings from preparative lEF of multiple PL enzymes of different pi’s.
The endoPL’s obtained from preparative lEF had similar characteristics, although
the cation preferences varied slightly. All but PL6 required cations for activity.
Most had dual preferences for Ca"^ and Co"^ ions, while PL2 strongly preferred Ca"^
to other cations. EndoPL’s are generally activated by Ca'^ ions (246), but little
information is available on their ability to use Co"^ ions. An endoPL of E. aroideae
was inhibited by Co"^ ions (141). The endoPL’s of Eca are similar to those of Ech
and PLII of Ecc 301, which could produce unsaturated dimers (99, 264, 312), unlike
the endoPL’s of EC 14, which produced unsaturated trimers as their final product (271).
The exoPL of pi 7.1 was found only in the periplasm. It is very similar to the
intracellular Ecc exoPL of pi 6.3 (271), although that enzyme was not tested for its
ability to use Co"^ ions. Hatanaka and Ozawa (122) isolated an exoPL that preferred
Co"*^ and Mn"^ ions as cofactors, although it was inhibited by EDTA. This enzyme
was isolated from the supernatant of cultures grown for 12 days and may have been
an intracellular enzyme released by autolysis.
Both PG’s were endo enzymes with similar characteristics, although their locations
differed: PGl was found only extracellularly, while PG2 was found in all fractions.
Both Eca and Ecc have been reported to produce an extracellular endoPG of a high pi 72
that has also been found intracellularly in Ecc (208, 271). Ecc and E, aroideae have been reported to produce an intracellular exoPG (120, 271), but the endoPG of pi 3.8 has not been reported previously. The endoPG’s of this strain of Eca differed from that of EC14, which could degrade trimer to monomer and had an optimum pH between 5.2 and 5.4 (208). The PG from other strains of Eca had a pH optimum of
5.0, although this value was obtained from assajdng crude enzyme (306).
The complexity of the extracellular pectic enzyme profile of Eca is similar to that of the other soft rot Erwinia. Ech is usually considered to produce five main extracellular endoPL’s of pi 4.6, 7.8, 8.3, 9.2, and 9.3 (PLa through PLe respectively)
(50). The genes for these enzymes have been cloned, indicated that they are truly isozymes (31, 155, 298). Additional endoPL’s can also be produced, possibly by post- translational modifications (51). It is not always possible to separate all of these enzymes by preparative lEF; for instance, PLb and PLc sometimes have one peak (51).
Ecc was originally considered to produce one extracellular endoPL (205), but research in recent years has identified multiple PL enzymes. Suguira et al. (279) have isolated two endoPL’s, while Tanabe et al. (280) have isolated four enzymes. Zink and
Chateijee (311) have isolated one extracellular endoPL and one extracellular exoPL, and Hatanaka et al. (119) have isolated a strain that produces only exoPL’s extracellularly. Ried and CoUmer (250) found that each strain of Ecc they examined produced three extracellular bands of PL activity that could be visualized by polygalacturonate overlays.
A published profile of the extracellular PL of Eca SR8 (8) shows 4 peaks of PL activity, instead of the 6 peaks of activity described here. This difference is due to the the size of the fractions collected from the lEF columns: 2.5 ml fractions were collected previously, while 1.25 ml fractions were collected in these studies. This 73
enabled the separation of 6 PL enzymes.
Eca had previously been shown to produce three extracellular endoPL’s, and the
large leading edge of the lEF profile suggests that it may contain additional enzymes
(239). The pTs of the enzymes are higher than those obtained from SR-8, but the
distribution of the peaks suggests that the enzymes are the same as those purified
from SR-8. The largest peak of pi 10.2 (CF-PGTE-2) is followed by a slightly smaUer
peak of pi 10.35 (CF-PGTE-3). These enzymes appear to be PL4 and PL3,
respectively. They are preceded by a small peak of pi 9.7 (CF-PGTE-1) that may be
analogous to PL5 or PL6.
It is difficult to compare the profile of Eca SR-8 to that of other organisms,
since enzyme production can vary between strains, and pi’s can vary greatly between
experiments. However, the complex of six extracellular PL’s produced by SR-8 invites
comparison with the PL profile of Ech. PL6 (pi 8.9) may be analogous to PLb of Ech.
The pi is higher than usual for this enzyme, but is similar to Keen and Tamaki’s
value of pi 8.8 for cloned PLb (147). Ecc produces a PL that is similar to PLb of
Ech. Roberts et al. (254) have cloned a gene from ECU that produces an endoPL of
pi 7.5 that does not macerate potato tissue, and Lei and Wilcox (171) have found
considerable homology between a pel gene from Ecc and Ech pelB.
If PL5 of Eca is analogous to PLb of Ech, PL6 may be equivalent to PLc.
Collmer et al. (51) have suggested that PLd may contain two enzymes not resolvable
by sucrose lEF, and it is possible that PL3 and PL4 may together comprise PLd. PLl
or PL2, or both, may be analogous to PLe. Eca does not produce a PL analogous to
PLa of Ech. Instead, it produces an endoPG of a similar pi. The soft rot Erwinia
are considered to produce only one extracellular PG (50); Eca is unique in its
production of two extracellular PG’s. 74
The cellular Icx^ations of these enzymes may provide some clues to their roles in pathogenesis. All of the extracellular pectic enzymes depolymerize NaPP in an endo manner. Both endoPL and endoPG have been found to macerate tissue (22, 50, 59).
The extracellular production of both types of enzymes may help to ensure the degradation of pectin in a variety of environments. The production of multiple forms of extracellular endoPL should confer an advantage to the pathogen. It may allow for a more complex regulatory strategy or provide for higher levels of PL (263). Some of the enzymes may be more stable than others under adverse conditions in the host
(35), and the enzymes may have differing abilities to damage host tissue. Pectin is a very complex polymer which is often interspersed with neutral sugars. The endoPL’s in Ech, Ecc, and Eca have been found to have varying substrate specificities (239,
263, 279, 280), which may aid in the degradation of such a complex substrate.
The endoPL’s of Ech have been found to have variable effects on plant tissue.
The alkaline PL’s appear to attack pectic polymers in a more random manner and seem more destructive to plant tissues than the neutral isozymes (50). There is strong evidence for their importance in soft rot pathogenesis (28, 215, 283).
The role of the neutral PL’s in pathogenesis is not as clear. They are "enriched for exo activity" and generate more unsaturated dimers than the other endoPL’s (99).
They tend to kill tissue at a faster rate than it is macerated and tend to be less effective at macerating than the alkaline enzymes (99). Mutants of Ech which lack these enzymes macerate potato tissue as effectively as the wild type (28, 257).
However, a mutant of Ech which lacked PLc took one week longer than the wild type to produce symptoms on Saintpaulia.
The ability of these enzymes to produce a large amount of unsaturated dimers and their toxicity to plant tissue suggests that they may have a role in inducing PL 75
synthesis during the initial stages of attack on plant tissue. If these enzymes serve to induce PL production in EcK they may also do so in Eca. If PL5 and PL6 are analogous to PLb and PLc, they are candidates for this function. It is interesting that the CF-PGTE-1 enzyme of Quantick et al. (239), possibly the same as PL5 or
PL6, was more toxic to potato tissue than the other enzymes isolated: it killed protoplasts more rapidly than it macerated. It was also the most thermolabile of the enzymes, and was quickly inactivated by chlorogenic acid.
If PL5 and PL6 are equivalent to PLb and PLc of Ech, the other extracellular endoPL’s should be more important than the other enzymes at macerating polygalacturonate during soft rot pathogenesis. CF-PGTE-3 was the only enzyme that could be isolated from extracts of rotted potatoes; it was much less affected by chlorogenic acid than the other PL’s. This enzyme is probably the same as PL3 from
SR-8.
In Eca SR-8, PLl, PL2, PL5, and PL6 are found only extracellularly. This
suggests that they have a role in the degradation of plant tissue, but not in the
catabolism of the breakdown products of pectin. PL3 and PL4 are also found
intracellularly, which may indicate that they play a role in catabolism.
The intracellular endoPL enzymes are located primarily in the periplasm. This is
in accordance with results from EcK in which most of the intracellular PL was
located in the periplasm (10, 282). However, studies with Ecc indicate that most of
the intracellular PL was in the cytoplasm (39, 97). Some of this disparity may reflect
differences in the growth stage of the cultures at the time of sampling (49).
The exoPL is located solely in the periplasm where it may facilitate the
production of unsaturated dimers from the oligouronides which have entered the cell.
This may be important in the induction of PL synthesis, since the breakdown products 76
of unsaturated dimers have been found to induce PL synthesis in Ecc and Ech (47,
289). The concentration of intracellular PL in the periplasm instead of the cytoplasm
should provide an advantage to the cell. It should facilitate the breakdown of
oligouronides to small fragments before they are transported into the cytoplasm. Such
small fragments would be readily available to the cell’s transport mechanism (60) for
the uptake of large macromolecules. A transport system for deoxyketuronic acids has
been described, although it is not known if larger oligouronides can be taken up (55).
The location of endoPLs in the periplasm may also facilitate their secretion to the
extracellular environment.
Once inside the cytoplasm, the fragments should be degraded to deoxyketuronic
acids and metabolized (49). In Ecc and Ech, unsaturated and saturated dimers are
degraded by oligogalacturonide lyase (OGL) to unsaturated monomer and D-
galacturonic acid. This enzyme may have been present in Eca, but may have been
inactivated by the high voltage used during isoelectric focusing (271).
The intracellular pectic enzymes of Eca differ from those of EC14 and Ech. EC14
produces three PL’s: endoPL of pi 9.4 (PDl), exoPL of pi 8.0 (PD2), and exoPL of pi
6.3 (PD3) (271). The intracellular endoPL’s of Eca (PL3 and PL4) have similar
characteristics to PDl, and the exoPL of Eca is very similar to PD3. However, Eca
does not produce an enzyme analogous to PD2, an exoPL which produces dimers and
trimers. A similar exoPL was also found extracellularly in Ecc 301 (311). Ech also
produces a similar profile of intracellular pectic enzymes. Three endoPL’s of pi 9.4,
9.0, and 7.8 have been reported (14), as has an exoPL (47).
EC 14 also produces intracellular endoPG of high pi, but in the isoelectric focusing
profiles from Eca, this enzyme was found only extracellularly. However,
chromatography of the periplasmic fraction revealed a large Nelson’s peak that eluted 77
in the void region and was much larger than the corresponding peak in the other fractions. The presence of such a large Nelson’s peak suggests that the activity is due to polygalacturonase, and the location of the peak in the void region suggests that the enzyme has a high pi.
The periplasm may contain a pre-secretory form of PGl that is unstable under the conditions of isoelectric focusing. After lEF, there were traces of PG with a much higher pi than that found extracellularly. Such a difference could be due to the presence of a highly basic signal sequence. An alternative, though less likely, possibility, is that PG2 had complexed with another protein to form a high pi
conglomerate with PG activity that could be separated by isoelectric focusing.
The pectic enzyme complex of Eca has been characterized from cells grown on
citrus polygalacturonate as their sole carbon source, and it is possible that there may
be differences in enzyme production when the cells are in contact with living plant
cells. Some enzymes and not others may be produced, or additional enzymes may be
induced. The substrate of citrus pectin may not be adequate to distinguish the more
destructive enzymes which are specialized for specific galacturonosyl linkages in the
plant cell wall (46).
It is also not clear whether all of the pectic enzymes are isozymes. By
definition, isozymes must be the products of different genes (204). Enzymes that are
the products of post-translational modifications cannot technically be considered
isozymes. The gene for PL4 has been cloned (8), but it is not clear whether the
other enzymes are produced from different genes. Some of the endoPL’s differ in
their ability to use cations and act at lower pH’s, suggesting that they have some
structural differences. Ech has five pel genes, and recent evidence indicates that
Eccll contains six pel genes and one peh gene (50), so it is probable that many of 78
the Eca enzymes are isozymes.
The localization and characterization of the pectic enzymes produced by Eca should facilitate further studies on the role of the individual enzymes in pathogenesis.
Ech has been said to have a more complex isozyme profile than Ecc (46), but the profile of Eca SR-8 rivals it in complexity. Chapter 3
THE EFFECT OF TEMPERATURE ON PECTATE LYASE
PRODUCTION
Introduction
Soft rot of potatoes is a destructive disease which is most commonly caused by species of Erwinia, Erwinia carotovora subsp. carotovora (£cc), Erwinia carotovora subsp. atroseptica (Eca), and Erwinia chrysanthemi (Ech). Environmental conditions determine which organism is associated with the disease, and temperature is of primary importance. Eca can grow at much lower temperatures than either Ecc or
Ech and is more of a problem in cool climates or in the cool seasons of warm regions.
These bacteria produce copious amounts of pectic enzymes which are necessary for the development of soft rot. Perombelon and Ghanekar (225) have presented evidence that differential production of pectic enzymes is responsible for the greater pathogenicity of Eca at cooler temperatures. They found that several strains of Eca produced large amounts of pectate lyase at 15"C and little or none at 30“C.
Pectate lyase production at the two temperatures was examined more closely by
determining the column profiles and cellular location of enzyme.
79 80
Methods
Cultural Conditions
The bacteria were grown aerobically in sodium polypectate minimal salts
(NaPPMS) broth at either 15"C or 30'C. The cultures at 15*C were grown on an
Orbit Gyrotory shaker at 150 RPM in an incubator, while those at 30‘C were grown in a Fisher Model 129 rotary water bath, shaking at approximately 90 rpm. 50 ml of
NaPPMS was inoculated with a loopful of bacteria from solid media and incubated until visible growth appeared, which was overnight at 30'C and usually after 2 days at
15*C.
To determine the patterns of growth at the two temperatures, 1 ml of the inoculum was added to 200 ml of media and aerated. Periodically, 3 ml samples were removed and their absorbance was measured at 600 nm. When the cultures were used
as a source of enzyme, 1 ml of the inoculum was added to each of 5 flasks of 200 ml of NaPPMS. The cultures were aerated until they reached early stationary phase, which occurred at an of 1.0-1.1 at 15*C and an of 0.75-0.85 at 30’C.
Enzyme Extraction, Purification, and Assays
The cultures were centrifuged, and the supernatant was used as the extracellular fraction. The periplasmic fractions were isolated by lysozyme-EDTA treatment (308), and the spheroplasts were lysed by sonification to release the cytoplasmic fraction as described in Chapter 2. The enzymes were precipitated with (NH^)2(SO)^ and purified by DEAE column chromatography (271). Pectate lyase was assayed by the TBA assay
(235), and the Nelson’s reducing sugar assay was used to determine polygalacturonase activity (209). 81
Polyacrylamide Gel Isoelectric Focusing
The crude enzyme preparations were subjected to isoelectric focusing on an ultrathin polyacrylamide gel (PAGIEF) as described by Ried and Collmer (249), except that the samples were applied in the middle of the gel and focusing was carried out at 6 W with a maximum of 2500 V. The PL enzymes were detected by a
NaPP-agarose overlay which was incubated at room temp for 11 hr and then stained for 20 min with 0.05% ruthenium red. 82
Results
Growth Curves
The temperature of incubation had a marked effect on both the growth rate and the growth yield of Eca in a sodium polypectate medium (Figure 10). At 15’C, the
bacteria rapidly reached an OD of 1.0 at 6(X) nm and had a prolonged stationary phase
before starting to decline. However, at 30“C, the bacteria reached their maximum
levels more slowly and had a less pronounced exponential phase. The cultures only reached an OD of 0.75-0.85, then started to decline fairly rapidly; there was no
distinct stationary phase. The cultures at 15‘’C reached their maximum level of
absorbance in 40 hours, while those at 30'C took over 70 hours to reach their peak.
Enzyme Activity
Quantitative measurements of the activities of the enzymes could not be obtained
from the crude preparations; the reaction mixtures usually had a precipitate which
altered the readings. Thus, the enzymes had to be purified further before precise
measurements of their activities could be determined.
The enzymes were purified by subjecting the crude protein to DEAE 53
chromatography. The amount of extracellular protein that needed to be applied to the
column to get clearly resolved peaks differed between the preparations from the two
temperatures. The protein from cultures grown at 15'C contained greater enzyme
activity than those grown at 30'C, and much less protein needed to be used from the
isolations at 15'C.
The extracellular column profiles from the two temperatures differed (Figure 11,
12). Each profile had two peaks of PL activity-a large peak that eluted in the void
and a smaller peak that eluted around 0.1 M NaCl gradient. The composition of the
second peak varied. In profiles from cultures at 30'C, it had a large peak of activity minimal saltsbroth at15*CandSO^C. Figure 10.Growth ofErwiniacarotovorasubsp.atroseptica inasodiumpolypectate
600 «600 0 20406080100120 Hours Hours 83 84
Figure 11. DEAE cellulose column chromatography of extracellular, periplasmic, and cytoplasmic pectic enzymes from Erwinia carotovora subsp. atroseptica incubated at 30'C. The (NH ) SO preparation was applied to a DE53 column, and enzyme was eluted by a stepwise gradient of 0-0.2 M NaCl in 0.05 M Tris-HCl buffer, pH 8.0. Fractions were incubated with 0.6% sodium polypectate for one hr and assayed by the TEA assay at A^^ (235) for pectate lyase activity (•) and by the Nelson’s r^ucing sugar assay at A^ (209) for polygalacturonase activity (o). Absorbance Absorbance Absorbance 2.0 0.5 1.5 1.0 0.0 0 10 20 30 Fraction Number 85 86
Figure 12. DEAE cellulose column chromatography of the extracellular, periplasmic, and cytoplasmic pectic enzymes of Erwinia carotovora subsp. atroseptica incubated at 15’C. The(NH) SO preparation was applied to a DE53 column, and enzyme was eluted by a step^use ^adient of 0-0.2 M NaCl in 0.05 M Tris-HCl buffer, pH 8.0. Fractions were incubated with 0.6% sodium polypectate for one hr and assayed by the TBA assay at A^^ (235) for pectate lyase activity (•) and by the Nelson’s reducing sugar assay at A^ (209) for polygalacturonase activity (o). Absorbance Absorbance Absorbance 2.0 1.5 1.0 0.5 0.0 2.0 1.5 0.5 0.0 1.0 2.0 1.5 0.5 1.0 0.0 0 NaCI Fraction Number 0.05 MTris-HCI 0.05 M0.10.2 30 88
as determined by the Nelson’s assay and a small peak of PL activity, as determined by the TBA assay. However, in cultures incubated at IS'C, this peak had a larger peak of PL activity than of Nelson’s.
The fractions within each peak were pooled and used to determine the specific activity of the PL in units/mg (Table 4). The PL activity from peak 1 was slightly higher at 15*C, but it was close to that at 30’C. However, the PL from peak 2 had a much higher specific activity at 15’C than that at 30*C: there was 64.92 units of activity at 15*C, while there were only 1.59 units at 30°C.
The total amount of PL activity in the pooled fractions from the columns was compared with the amount of protein applied to the columns and used to calculate the total units of PL found in the extracellular fractions. This method gives an approximation of the total PL, since the small amounts of PL activity found between the peaks of the column are not included in the total. The extracellular fraction of the cultures from 15"C contained over ten times the amount of PL found in the extracellular fraction of the cultures from 30’C (Table 5).
Similar calculations on the periplasmic and cytoplasmic fractions enabled the distribution of PL between the subcellular fractions to be determined. About 95% of
the PL from cells grown at 15’C was found extracellularly, while only 54.9% of the PL
from cells incubated at 30*0 was in the extracellular fraction (Table 6).
The composition of the periplasmic fraction also varied between cells from 15*C
and 30’C (Figure 11, 12). The column profiles from 30'C had a large peak of PL
activity in the void region, followed by a smaller, but substantial PL peak around 0.1
M NaCl gradient. Both peaks also included large peaks of Nelson’s activity. Peak 1
from 15*C contained much less PL activity and had a small Nelson’s peak, even
though more protein had been applied to the 15*C columns. The second peak in the 89
Table 4. The specific activity of pectate lyase peaks from DEAE column chromatography of Erwinia carotovora subsp. atroseptica incubated at 15*C and 30‘C.
Specific activity* 30'C lyc
Extracellular Peak 1 19.01 23.48
Peak 2 1.59 64.92
Periplasmic Peak 1 53.87 24.03
Peak 2 17.44 5.03
Cytoplasmic Peak 1 20.30 28.71
Peak 2 1.93 4.42
‘Specific activity is given in units/mg of protein. The fractions in each peak were pooled, and 100 |xl of enzyme was incubated with 0.6 % sodium polypectate and assayed by the TEA assay (235). One unit is equivalent to one millimole of D- galacturonic acid formed per hour. Protein content was determined by the Bradford assay. 90
Table 5. The units of pectate lyase activity in the subcellular fractions of Erwinia carotovora subsp. atroseptica incubated at 15*C and 30"C.
Pectate lyase activity^
30"C 15"C
Extracellular Peak 1 320.35 2044.75
Peak 2 19.08 1654.29
Total 339.43 3699.04
Periplasmic Peak 1 148.89 92.07
Peak 2 41.06 12.00
Total 189.95 104.07
Cytoplasmic Peak 1 25.77 63.65
Peak 2 1.36 9.79
Total 27.13 73.44
^Pectate lyase activity was determined by the TEA assay (235). The total amount of activity in each peak is given in units. One unit is equivalent to one millimole of D- galacturonic acid formed per hour. 91
Table 6. The distribution of pectate lyase activity in the subcellular fractions of Erwinia carotovora subsp. atroseptica grown at 15*C and 30°C.*
Percent of Pectate Lvase Activity'’
Temperature Extracellular Periplasmic Cvtoplasmic
30“C 54.9 39.4 5.6
15*C 95.4 2.9 1.9
“The supernatant was removed by centrifugation, and the periplasmic fraction was isolated by a modified lysozyme-EDTA treatment (308), followed by centrifugation. The spheroplasts were then lysed by sonification to release the cytoplasmic fraction.
'’Pectate lyase activity was determined by the TBA assay (235). 92
15*0 columns contained very small Nelson’s and PL peaks and was located in the void region. This peak may not have been equivalent to peak 2 from the 30“ C columns, which was found around 0.1 M NaCl gradient.
The specific activity of the PL in peaks 1 and 2 was greater from cultures grown at 30“C than those grown at 15*C, and the periplasmic fraction from cells grown at
30“C contained more units of PL activity than those at 15"C. 39% of the cells* PL was found in the periplasm at 30“C, while only 2.9% was found in the periplasm of cells at 15“C.
The profiles of the cytoplasmic fractions were similar at 15“C and 30“C. Both had a large peak of PL activity in the void, and a small peak of PL activity in 0.05
M-0.1 M NaCl gradient. In addition, there was a small peak of Nelson’s activity above the small PL peak. The 15“C profiles had an additional small PL and Nelson’s peak in the void after the large PL peak. The specific activity of the PL of both peaks was higher at 15“C than at 30“C, although a smaller percentage of the cells’
PL was in the cytoplasm at 15“C than at 30“C.
Polyacrylamide Gel Isoelectric Focusing
Each of the subcellular fractions contained multiple bands of PL activity (Figure
13). In this particular overlay, the periplasmic and cytoplasmic fractions differ in the production of the uppermost band of PL activity. It is present in the cytoplasmic fraction at 30“C and absent from the periplasmic fraction, while the reverse is true at
15“C. However, the overlays are very variable, and differences between the temperatures were not produced consistently. Overall, the same PL bands were present at the two temperatures. 93
SO’C 15‘C ABC ABC
Figure 13. Isoelectric focusing on a polyacrylamide gel of extracellular (A), periplasmic (B), and cytoplasmic (C) pectate lyase enzymes of Erwinia carotovora subsp. atroseptica incubated at IS'C and 30*C. The enzymes were detected by placing a pectate-agarose overlay was over the gel for 11 hr and staining with 0.05% ruthenium red for 20 min. 94
Discussion
The bacteria grown at 15*C and at 30"C differed in their patterns of growth and their production of pectic enzymes. The cultures at 15"C quickly reached an of
1.0 and had a prolonged stationary phase before declining, while those at 30*C took longer to reach an absorbance of 0.75-0.85 and started declining immediately. There was no distinct stationary phase in the cultures at 30*C.
Perombelon et al. (226) found similar differences in 30* C cultures incubated at different temperatures. They examined the growth of Eca and Ecc populations in wounded potato lenticels incubated anaerobically at 16*C and 22*C. At 22*C, the populations increased and decreased in a manner similar to the Eca cultures that were grown in sodium polypectate at 30° C. The bacteria reached lower population levels at
22*C than at 16°C and started declining rapidly, while the populations at 16“C peaked at higher levels and remained relatively constant.
Campos et aL (36) examined the growth of Ecc and Eca in a casamino acids- peptone-glucose broth medium at various temperatures. The cultures were not aerated and presumably were anaerobic. They also found that cultures at the lower temperatures of 12*C and 16°C, reached higher absorbance levels than those at the higher temperatures of 24*C, 28°C, 32°C, and 36*C. However, the cultures incubated at the warmer temperatures had a prolonged stationary phase, unlike the bacteria grown in the potato lenticels or in sodium polypectate broth medium.
Because of the different patterns of growth at 15°C and 30°C, it was difficult to isolate enz5m[ies from cultures which were at exactly the same stage of growth. The prolonged stationary phase of the cultures at 15°C made it easy to isolate enzymes from this phase. However, the cultures at 30°C started declining quickly after they reached the level of maximum growth. It was necessary to isolate the enzymes before 95
the cells started lysing in decline phase, and with no clear stationary phase, the cells may still have been in late log phase when the enzymes were isolated.
Collmer and Bateman (52) have reported a dramatic increase in the level of extracellular PL during the transition from logarithmic to stationary phase in Ech.
This was also accompanied by a wave of intracellular PL activity. If a similar situation occurs in Eca, the difference in enzyme production and distribution at 15*C and SO'C may reflect a differential secretion by cultures at different growth phases.
Isoelectric focusing on PAGE gels indicated that the PL profile was similar at the two temperatures, however the specific activity and the amount of PL found extracellularly were much greater at 15’C than at 30*C, and over 95% of the PL of the cells at 15*C was found extracellularly. Only 2.9% of the cells’ PL was contained in the periplasm. However, in the cells grown at 30“C, 39.4% of the PL was found in the periplasm, and only 54.9% of the PL was extracellular. This difference could be due to secretion of the PL from the periplasm to the cell’s exterior. If the cells at
30°C were still in logarithmic phase, they may not yet have secreted as much PL as they would have by the end of stationary phase.
In addition to the differences in the distribution of the PL activity between the subcellular fractions, there are also differences in the DEAE chromatography profiles of enzymes from the two temperatures. While the exact composition of the enzymes in the peaks cannot be determined without subjecting them to isoelectric focusing, some differences are clearly evident. The Nelson’s peak in peak 2 is much smaller in the extracellular profile from 15*C than that at 30*C. This probably indicates that less polygalacturonase is present extracellularly during the stationary phase of the cultures at 15*C. Preparative lEF of the extracellular enzymes from 30*C had revealed a PG with a pi of 3.8 (Chapter 2); this may be the enzyme found in the 96
second peak.
The profiles of the periplasmic fractions are quite different between the cultures from the two temperatures. In peak 1 of the 15‘C profile, the Nelson’s peak is much smaller than that from the 30“C profile. This may indicate that polygalacturonase present in the periplasm at 30*C has been secreted at 15"C. However, the low amount of extracellular Nelson’s activity at 15"C argues against this. It is also possible that the composition of the PL in peak 1 is different at the two temperatures. The enzymes at 30* C may have activity under the conditions of the
Nelson’s assay, while the ones at 15*C may not have this sort of activity.
The periplasmic profile from 30*C also has a second peak with equally large PL and Nelson’s peaks. lEF of the periplasmic fraction from 30*C has shown the presence of an exoPL with noticeable activity under the Nelson’s assay (Chapter 2).
This enzyme has a pi of 7.1 and would probably require a greater concentration of
NaCl to be eluted than the other PL’s, because of its low pi. It is very probable that the second peak in this profile is primarily composed of this exoPL. The profile from the periplasmic fraction of 15*C has a second very small peak that elutes in the void region and may not be equivalent to the second peak from the 30*C profile. This suggests that the exoPL may not be found in large amounts in the periplasm of the cells isolated in stationary phase at 15*C.
The exoPL produces unsaturated dimers, and these products are important in inducing PL production in Ecc and Ech (48, 289). The location of this exoPL in the periplasm would facilitate the generation of unsaturated dimers from oligomers which have been transported into the cells. Cells which are in stationary phase probably have already been induced to produce their inducible enzymes, and it is possible that such cells would no longer require an exoPL for induction. An alternative possibility 97
is that the exoPL has been secreted to the extracellular environment and may
represent the slightly larger second extracellular PL peak found at 15*C, although this peak has very little Nelson’s activity.
The profiles of the cytoplasmic fraction are very similar in preparations from
IS^C and 30“C. In E. colU most proteins destined for secretion are transported to
the periplasm as they are synthesized (241). If is this is also true in Ecfl, the only
enzymes expected to be found in the cytoplasm would be those with catabolic
functions. These enzymes probably do not vary at different temperatures. The
specific activity of the PL in the cytoplasm is higher at 15*C. This may not reflect
the situation in vivo, since the isolation procedures resulted in cytoplasmic
preparations varying greatly in their protein content. Sometimes portions of the
cellular pellet ended up in the soluble cytoplasmic fraction.
It is not clear how applicable these differences in pectic enzyme production are
to the pathogenicity of Eca at 15*C and SO'C. P^rombelon and Ghanekar (225) have
suggested that differential pectate lyase production at 15*C and 30*C may be
responsible for the increased pathogenicity at 15“C. They found that several strains
of Eca produced little or no extracellular PL at 30“C, while producing substantial
amounts at 15“C. The results described here contradict their findings. While Eca
SR-8 produced more extracellular PL at 15°C, it still produced substantial amounts at
30‘C.
Because of the difficulties in isolating enzyme from the exact same growth phase
of the bacteria at the two temperatures, it is not clear whether the differences in
enzyme production were due to the differences in temperature or to the differences in
stage of growth at which the enzymes were isolated. Chapter 4
PARTIAL PURIFICATION AND CHARACTERIZATION OF
EXTRACELLULAR PROTEASE
Introduction
Several species of Erwinia are destructive pathogens of an array of plants, and they produce a variety of extracellular degradative enzymes that facilitate their pathogenesis. Pectate lyase has been the most studied of these enzymes, but polygalacturonase, cellulase, and protease may all have roles in pathogenesis.
Protein is an important structural component of the plant cell wall and membrane and plays a role in some plants’ induced resistance to pathogens (187). It would be advantageous for pathogens to degrade protein during pathogenesis, and extracellular proteases would facilitate this. The proteases from Erwinia chrysanthemi (Ech),
Erwinia carotovora subsp. carotovora (Ecc), and Erwinia amylovora have been studied, but there is no information on protease production in Erwinia carotovora subsp. atroseptica (Eca) (205, 264, 268, 287, 304). I report here the partial purification and characterization of an extracellular protease from Eca strain SR-8.
9,8 99
Methods
Cultural Conditions
Erwinia carotovora subsp. atroseptica strain SR-8 was obtained from A. Kelman,
Univ. of Wisconsin. The bacteria were cultured for enzyme extraction in broth containing 0.5% sodium polypectate and minimal salts (NaPPMS) (271). The bacteria were grown at 15*C overnight on a gyratory shaker (150 RPM). A one ml sample was transferred to 100 ml of NaPPMS and grown until early stationary phase was reached.
The bacteria were maintained on NaPPMS agar at 4“C.
Enzyme Extraction and Purification
The extracellular fraction was isolated by centrifuging the cultures at 20,000 g at
4'’C for 20 min. The supernatant was concentrated with Amicon filters and then stored at 4oC. 1 ml of the crude preparation was applied to a 1.6 x 16 cm diethylaminoethyl (DEAE 53) cellulose column as described in Stack et al (271). 3 ml fractions were collected and assayed.
Enzyme Assay
A modification of the method used by Tseng and Mount (287) was used to assay
for protease activity. 1(X) }il of enzyme was added to 100 |il of substrate consisting of
1% Bacto gelatin (Difco) in 0.05 M Tris-Mal buffer, pH 7.5. The fractions were
incubated at 30“C for 12 hr when assaying columns and for 4 hr when characterizing
the enzymes. After incubation, 600 p.1 of 20% trichloroacetic acid (TCA) was added.
After 30 min, the mixtures were centrifuged at 5000 g for 20 min. at 19'C using an
SM64 rotor. The supernatant contained TCA-soluble hydrolysis products, while
undegraded gelatin was precipitated by the TCA and remained in the pellet. The
amount of protease activity was determined by measuring the absorbance of the
supernatant at 280 nm against a reaction blank containing water instead of enzyme. 100
Characterization of Enzyme
Purified protease from the DEAE column was dialyzed overnight in several liters of dH20 and used to determine the effect of ethylenediaminetetraacetic acid (EDTA) on the enzyme. Varying amounts of EDTA were tested in triplicate. 300 |xl of enzyme was incubated with EDTA for 30 min. in a reaction volume of 335 |il. 100 pi of enzyme was removed, added to 100 pi of substrate, and assayed as described above.
Enzyme was also preincubated with water as a control.
The effect of cations on enzyme activity was also examined. Enzyme was
preincubated with 2.5 mM EDTA for 30 min, and 100 pi was removed and added to
100 ul of substrate containing 10 mM of various cations. The optimal amount of
cation that had to be added to restore activity was determined by adding 100 pi of
inactivated enzyme to 100 pi of substrate containing increasing amounts of cation.
Polyacrylamide Gel Isoelectric Focusing
The crude enzyme preparation was subjected to isoelectric focusing on an
ultrathin polyacrylamide gel (PAGIEF) as described by Ried and Collmer (249), except
that the samples were applied at the cathode and focusing was carried out at 6 W
with a maximum of 2500 V. 9.4 pg of the extracellular protein was applied to the gel
after pre-electrophoresis, and protein standards supplied by BioRad were also applied.
After electrophoreses was completed, an agarose-substrate overlay containing 1%
gelatin in 0.05 M Tris-Mal, pH 7.5, was placed on top of the gel and incubated at
room temperature for 18 hr. The overlay was placed in 10% TCA until bands of
protease activity appeared in the overlay and stained for 45 min with 2% Coomassie
Brilliant Blue in methanol/water/glacial acetic acid (50/50/10). The overlay was
destained with methanol/water/glacial acetic acid (35/66/10) for 2 hr. 101
Results
Enzyme Purification
F*rotease activity was detected in the crude extracts, but these extracts were difficult to assay. Often, the samples had a high content of protein that prevented an accurate determination of the protease activity of these extracts. The samples were further purified by chromatography which resulted in a large peak of activity that eluted with 0.3-0.4 M NaCl (Figure 14).
Effect of EDTA and Reactivation by Metal Ions
The enzyme is inactivated by EDTA in concentrations of 2.5 mM or greater
(Figure 15). Several divalent cations restored activity to enzyme which had been inhibited by EDTA. Co"^ and Mn"^ ions were the most effective at restoring activity, but Zn^ and Ca"^ ions were also effective (Table 7). Na^, Mg'^, and Ba^ ions were ineffective at restoring activity. The optimal amount of Co'^ ions that restored
activity was 10 mM (Figure 16). Greater amounts of the cation were less effective at restoring activity. Similar results were found with Mn"^ ions.
Polyacrylamide Gel Isoelectric Focusing
Overlays containing gelatin revealed one band of protease activity (Figure 17).
This band was below the lowest pi marker on the gel, phycocyanin, which had a pi of
4.65. This indicates that the protease had a pi of less than 4.65. Overlays of gelatin
at pH 9.75 and pH 4.0 also revealed one band of activity at the same position,
indicating that the protease had activity over a broad pH range. 102
Fraction Number
Figure 14. DEAE cellulose chromatography of extracellular protease. The (NH^)2SO^ preparation was applied to a DE53 column, and enzyme was eluted by a stepwise gradient of 0-0.4 M NaCl in 0.05 M Tris-HCl buffer, pH 8.0. Fractions were incubated for 12 hr with 1% gelatin in 0.05 M Tris-Mal buffer, pH 7.0, and assayed as described. Figure 15.Effect of EDTAonproteaseactivity.Purified enzymewasincubatedwith EDTA at30“C.After 30min,100p-lsampleswereremoved andincubatedwith1% gelatin in0.05MTris-Mal, pH7.5,for4hrandassayed asdescribed. 280 0.04 0.03 0.02 0.01 0.00 EDTA (mM) -f-«- 10 12 103 104
Table 7. Restoration of protease activity by metal ions/
Ion Reactivat
CoCl 2 156 MnCl^ 125
ZnSO, 109
CaCl, 93
NaCl, 7
MgCl^ 0
BaCl, 0
EDTA 7
‘Purified enzyme was incubated for 30 min. at 30°C with 2.5 mM EDTA. 100 fil samples were then assayed in the presence of 10 mM metal ions at pH 7.5 as described.
‘The values given are the average of three determinations, normalized to the value for untreated enzyme. 105
Figure 16. Reactivation by Co^ ions of protease inhibited by EDTA. Purified enzyme was incubated with 2.5 mM EDTA at 30“C for 30 min. 100 ^il s^ples were removed and incubated with 1% gelatin in 0.05 M Tris-Mal, pH 7.5, containing CoCl^ for 4 hr and assayed as described. Each value is the average of three determinations. 106
Figure 17. Isoelectric focusing of extracellular protease on a polyacrylamide gel. The enzymes were detected by a gelatin-agarose overlay which was placed over the gel for 18 hr, placed in 20% trichloroacetic acid for 30 min, stained with 1% Coomassie Brilliant Blue for 45 min, and destained for 2 hr as described. There is one band of enzyme activity, which is indicated by the arrow. 107
Discussion
Erwinia carotovora subsp. atroseptica strain SR-8 was found to produce a single band of extracellular protease activity that was visualized by gelatin overlays of
PAGIEF gels. The protease could be inactivated by EDTA and reactivated by cations, suggesting that it is in the class of metal chelator sensitive proteases (180). Co^ and Mn"*^ ions were the most effective, but Zn"^ and Ca"^ ions also restored some activity. The cation preferences of the Eca protease are similar to those of some other bacterial proteases that are sensitive to metal chelators. Ca"^, Co'*'"*', Zn"^, and
Mn'*^ restored activity to the inactivated protease of Xanthomonas alfalfae (243).
Bacillus subtilis (288) and Escherichia coli (42) produce proteases that contain Zn'*^ and can be reactivated by Zn'^, Co*^, or Mn'^ ions. Proteases from many species of
Pseudomonas can be reactivated by Co"*^, Zn"*^, Mn"*^, and Ca"*^ ions (84). The enzyme from Eca is inhibited by large amounts of cations; this has been reported for other proteases from this class (84).
The protease produced by Eca appears to be different from the extracellular
protease produced by Ecc. Protease from Ecc EC91 eluted in the 0.3-0.4 M NaCl
gradients of the DEAE columns, but it had a pi of 4.95 and was not inhibited by
EDTA (268). Lack of inhibition by EDTA suggests that the enzyme is not in the class
of metal chelator-sensitive proteases. This enzyme is probably the same as that
previously described by Mount et al (205) as eluting in the 0.4 M NaCl gradient. In
addition, a protease which eluted in the void volume and had a pi of 8.3 has also
been described (287). This enzyme may have been an intracellular enzyme released by
autolysis, since the cultures were grown for 14 days before the enzyme was isolated.
Erwinia chrysanthemi and Erwinia amylovora also produce extracellular proteases.
Two extracellular proteases were isolated from a hyperproteolytic mutant of Ech 108
(304). One had a pi of 4.6 and was produced in low amounts by the wild-type strain.
The other enzyme was a serine protease with a pi of 5.8. This protease seemed to be rapidly degraded in the wild-type strain, and was antigenically unrelated to the other protease.
Erwinia amylovora produces two extracellular proteases. Protease n is a metal chelator sensitive neutral protease with a pi of 4.6. It was produced in culture and in the ooze from immature pear fruits infected with the bacterium (264). Protease I had a pi of 9.9 and was only detected in the ooze. This enzyme was also inhibited by EDTA.
It is interesting that two other species of Erwinia produce proteases with a pi of
4.6. The enzyme from Ech was not characterized, but Protease 11 of E. amylovora has
characteristics similar to the Eca protease. It is not clear why Ecc and £cfl, two
closely related subspecies, produce proteases that are in different classes, although
there are several possibilities.
One such possibility is that the production of protease is affected by the type of
host. Garibaldi and Bateman (100) have found that strains of Ech from
chrysanthemum and Diejfenbachia produced large amounts of extracellular protease,
while those from com and carnation did not. It is not known if these differences are
quantitative or qualitative. It is possible that some hosts induce the production of a
different protease.
Also, the structure of the cell wall protein from dicots and monocots is very
different: the protein in dicots is very rich in hydroxyproline that is highly
glycosylated, making it very resistant to proteolytic degradation (160). In contrast,
the protein of monocots contains very little hydroxyproline, and the hydroxyproline
that is present has a low amount of glycosylation. This may enable the cell wall 109
protein of monocots to be more easily degraded. Ecc EC14 was originally isolated from calla lily (205), while Eca SR-8 was isolated from potato (226). Perhaps strains from monocots produce different protease than that from dicots.
Potato tubers produce many different protease inhibitors, and inhibitors I and H, in particular, are stored in high concentrations in the tubers (247, 261). These compounds are potent inhibitors of serine proteases and have been correlated with resistance to Colorado potato beetles. Unlike Ecc and Echy Eca produces a metal- chelator sensitive protease. Also, unlike the other soft rot Erwinia, Eca is limited almost entirely to potatoes (227). It is conceivable that Eca may produce a non¬
serine protease to avoid the serine protease inhibitors. The strains of Erwinia that have been shown to produce serine proteases were not isolated from potatoes.
Another possibility is that Erwinia generally produces two proteases, one of which activates the other. Such a situation exists in Pseudomonas aeruginosa: one protease
accumulates in the periplasm and is secreted as an inactive precursor. It is then
converted to active protease by the action of a second, distinct protease (140). Ech
has been shown to produce two proteases, and it is possible that Eca and Ecc may do
this under some conditions. The protease of one may have been the activated form of
the precursor, while the other may have been the enzyme to cleave the precursor,
which had not yet been released. There is no experimental evidence to suggest this.
The role of proteases in the pathogenesis of the soft rot Erwinia species has not
been clearly determined. Friedman (92) found more virulent strains of Ecc to produce
more protease than avirulent strains, but protease from Ecc did not macerate potato
or cucumber tissue (205), although it did cause cucumber protoplasts to burst (287).
This suggests that it may have some effects on the plant’s membrane. Although the
HRGP in the primary cell wall is very resistant to proteolytic degradation, the middle no
lamella of potatoes contains a protein component that is not rich in hydroxyproline
(138,160). It is possible that this protein could be more easily degraded than the primary cell wall protein and could be degraded by Erwinia protease.
The degradation of structural cell wall protein during the early stages of infection would be expected to facilitate cell wall breakdown and, thus pathogenesis, but this does not appear to happen. In addition to the insoluble cell wall protein, potatoes and other Solanaceous plants contain soluble hydroxyproline-rich glycoproteins
(HRGP) that agglutinate avirulent strains of P. solanacearwn (265). These proteins are capable of agglutinating other kinds of gram-negative bacteria, but did not agglutinate most strains of Erwinia tested. If these proteins affected soft rot pathogenesis, their degradation would facilitate the process of pathogenesis, but agglutination was not related to the pathogenicity of the bacteria on potato (102).
The protease of Ecc and Eca was produced by cultures growing on sodium polypectate as a sole carbon source, and Ecc cultures grown in LB broth did not produce the enzyme (F. Smith, unpublished). Since the breakdown products of NaPP induce other enzymes that are involved in pathogenesis, the production of protease by bacteria grown on this medium may indicate that the enzyme is involved in pathogenesis. The Ech proteases were not produced in minimal media, but were induced by LB medium containing tryptone or casein hydrosylates (304). The differential production may also be due to a general regulatory phenomenon unrelated to pathogenesis. Some extracellular proteases are subject to catabolite repression, which does not necessarily involve cAMP (84). For instance, the extracellular proteases of Bacillus are constitutive, but are strongly repressed by complex growth media and amino acids (236). The Erwinia proteases could also be regulated in such a manner. Ill
Protease secretion in Ech proceeds normally in mutants unable to secrete pectate
lyase, polygalacturonase, or cellulase (10, 282). Also, E. coli clones, which contained
both pectate lyase and protease genes from Ech, secreted protease while retaining
pectate lyase in the periplasm (16, 282). This suggests that a different pathway of
secretion, and possibly, a different mode of regulation, is involved for protease in this
organism. This may indicate that the protease has a different role in the organism’s
survival than that of the cell wall degrading enzymes.
The pectolytic and cellulolytic enzymes facilitate the invasion and degradation of
plants by degrading the components of the cell walls, particularly the pectic
substances. It is possible to macerate plant tissue and kill cells with pure pectic
enzymes; thus protease is not essential for this process. However, extracellular protease may serve to produce nutrients for the bacteria. This would be useful in
diseased tissue and also in the saprophytic survival of the bacteria, particularly in
soil. The availability of nitrogen in the soil is limited, and plant materials rich in protein are metabolized more readily than those that are not (6). The breakdown of protein provides both carbon and nitrogen for microorganisms, and the production of extracellular protease during a saprophytic phase in the soil should enhance the
survival of the bacteria. APPENDIX
Stock solutions for Nelson’s reducing sugar assay
Solution A:
25 g anhydrous sodium carbonate
25 g Rochelle salts
25 g sodium bicarbonate
200 g anhydrous sodium sulfate,
made to 1 liter with H^O
Solution B:
a 15% solution of cupric sulfate pentahydrate in water slightly acidified
with H^SO^
Solution C:
25 parts of reagent A plus 1 part of Reagent B made just before use
Solution D:
25 g ammonium molybdate
21 ml of 96% sulfuric acid
3 g sodium monohydrogen orthoarsenate heptahydrate
made to 500 ml with H^O
incubate for 24 hr at 37“C and store in brown, glass stopper bottle
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