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Habitats

The lysobacters live in soil and fresh water and appear to be wide spread and in many places are rather abundant. They have been isolated from soils in the Netherlands (Veldkamp, 1955), Canada (Christensen and Cook, 1978; Katznelson et al., 1964; Peterson et al., 1966), the United States (Ensign and Wolfe, 1965), and Scotland (Daft et al., 1975). In Scotland, they have been found in agricultural soils and sand dunes where the pH was neutral to slightly alkaline (pH 8.8) and not below pH 6. Population densities of up to 500 plaque-forming-units (PFU) per cm3 were observed. This may seem very low, but the isolation method was designed only to detect organisms that lyse cyanobacteria (forming plaques in the cyanobacterial lawns), which may not be the case with all lysobacters. Also, the counts would be much reduced if most of the lysobacters are attached to particles. In soil, the lysobacters probably subsist by degrading various biomacromolecules and microorganisms other than cyanobacteria. In fact, lysobacters are known to decompose nematodes and different kinds of bacteria, as already mentioned. So, while their ecological niche is not really identified, the enzymatic equipment and lytic capabilities of the lysobacters suggest that they preferentially live in places rich in (recalcitrant) organic matter and microbial life.

Much more is known about the freshwater habitats of the lysobacters because there strains exist that can lyse living and healthy cyanobacteria and for this reason aroused much interest (see reviews by Stewart and Daft, 1976, 1977). The bacteria were found in fish ponds in Israel (Shilo, 1967; Shilo, 1970), in sewage plants in the United States (Stewart and Brown, 1969, 1971) and Scotland (Daft et al., 1975), and in lakes, reservoirs, and rivers in Great Britain (Daft et al., 1973, 1975). This, of course, does not mean that those lytic strains are the only lysobacters living in fresh water, as lysobacters were also isolated from that source by other techniques, and the ability of these strains to lyse cyanobacteria was never specifically demonstrated (in Canada: Christensen and Cook, 1978; in Germany: H. Reichenbach and coworkers, unpublished observations).

In contrast to cyanobacterial viruses, the lytic lysobacters have a wide activity spectrum with respect to the cyanobacterial species attacked. Thus, strain FP-1 lysed 9 out of 11 species of unicellular and filamentous cyanobacteria whereas the green alga Chlorella pyrenoidosa and the chrysophyte Prymnesium parvum were completely resistant (Shilo, 1970). All Gram-negative bacteria tested were also lysed, but Gram-positive bacteria were not. Strain CP-15 disintegrated 21 out of 23 strains of cyanobacteria (Daft et al., 1973), and strains CP-1 to -4 among themselves lysed 29 out of 42 cyanobacteria from nine different genera (Daft and Stewart, 1971). In the latter case, there were slight differences among the four strains with respect to the spectra of cyanobacteria attacked, and also different strains of the same cyanobacterial species could vary in their sensitivity to lysis. All 16 British Lysobacter strains used in these studies were serologically related and may have belonged to one species (Stewart and Daft, 1976).

The population densities of lysobacters observed in British waters are usually low (Daft et al., 1975), but since lysobacters attach themselves to plankton and other particles, the counts may not reflect the true numbers. In lakes in summer and at the water surface, between 0 and 400 PFU/ml were found, and the numbers decreased rapidly with depth (to 1 m). But 94% of all samples contained lytic bacteria. At the height of cyanobacterial water blooms, 0.001 to 0.05% of all cultivatable bacteria were lytic, and in general the population densities of cyanobacteria and lytic bacteria correlated well, with a slight shift in the time of the maxima. Large differences in the cell numbers could occur in simultaneous samples taken from different sites in the same lake. This apparently was simply due to wind drift which caused the plankton to accumulate at certain places. In samples with particularly high densities of cyanobacteria, the numbers of lysobacters could drop substantially. As the lysobacters are strict aerobes, their decline in number under such conditions was explained by oxygen depletion during the night, but perhaps the bacteria only seem to disappear because there was now an increased opportunity for attachment. In February, after 3 months of low phytoplankton density (and low temperature), the count of lytic bacteria fell to zero at the water surface, but some lysobacters still survived in the depth at the sediment-water interface. From there and from soils around the lakes, the repopulation of the water bodies may take place in spring. Another source for lytic bacteria was found in sewage works where in summer the counts in the final effluents were around 700 PFU/ml, and in the effluents from filter beds even as high as 1,300 PFU/ml. Also, in sewage plants, the numbers dropped during winter, in February to about 1% of the summer counts (Daft et al., 1975). Dense populations of lytic bacteria were found in the sand filters of a waterworks at a reservoir with blooming cyanobacteria. Up to 2,500 PFU/ml were found in the effluent, and 59,000 PFU/ml on the filter sand. The maximum in the open reservoir was 92 PFU/ml (Daft et al., 1973). No lysobacters were detected in water from underground springs (Daft et al., 1975).

While the lysobacters are able to lyse bloom-forming cyanobacteria, their low population densities make it questionable whether they really play a role in the control of natural water blooms. In field trials, at least 105 cells/ml were required to induce rapid lysis in cyanobacterial populations. In small bays at the edge of a reservoir, which were separated artifically from the main body and inoculated to a density of 106 bacteria/ml, there was clear evidence for lysis within 24 h at 13°C, and within 60 h, the Microcystis population was completely destroyed. In the lysing cell suspension the number of lytic bacteria rapidly declined, which again was explained by a lack of oxygen (Daft et al., 1973; 1975). In summary, it appears that cyanobacteria and lysobacters have similar growth requirements and simply coexist rather than prey upon one another. As the lysobacters can grow perfectly well as saprophytes, they may utilize material, including biomacromolecules, excreted by the cyanobacteria. In fact, they appear to interfere in a subtle way with the photosynthesis of the cyanobacteria and their excretion of dissolved organic carbon, reducing the rate of the former and stimulating the rate of the latter, and are able to rapidly assimilate excreted material, particularly certain amino acids, and to grow exclusively on it with a generation time of about 10 hrs (20°C). In a similar way they also exploit bloom-forming green algae like Scenedesmus quadricauda, which they cannot lyse at all (Fallowfield and Daft, 1988). Under conditions unfavorable for the cyanobacteria, the lysobacters also may lyse some of them, but they probably have little to do with the cyclic break down of water blooms in nature.

Isolation

Two properties of the lysobacters may be used for their enrichment: their efficient hydrolytic exoenzymes and their gliding motility. As both attributes are also found with other organisms, the enrichment techniques are not entirely specific.

Christensen and Cook (1978) recommend enriching soil samples with chitin, ground mushrooms, or Arthrobacter cells, and then leaving them for at least one month. After this period, the soil is suspended in water and appropriate dilutions are spread on yeast agar.

Yeast Agar

Baker’s yeast

0.5%

Agar

1.5%

Adjust to pH 7.2 and autoclave.

After incubation, colonies arise that are surrounded by lysis zones, and their number increases substantially during the enrichment phase. Two types are usually observed: 1) pink-colored (Lysobacter antibioticus) and 2) cream-colored (L. enzymogenes). Rarely, also, an off-white gummy colony may appear (L. gummosus). On subcultivation, most of the cream colonies produce two colony variants: 1) dirty-white mucoid, and 2) yellowish nonmucoid. When water samples from lakes and rivers are used, yellow-brown colonies are obtained (L. brunescens).

As all lysobacters appear to degrade chitin (Christensen, 1989), one also could try to isolate them on chitin agar as described by Veldkamp (1955). He used 1% of finely powdered chitin suspended in a medium containing 0.1% K2HPO4, 0.1% MgSO4, and 2% agar. He purified the chitin from shrimp shells by a very cumbersome procedure, so a simpler method is given here as well as two chitin media, which are used in our laboratory with excellent results in enriching for cytophagas and myxobacteria.

Preparation of the Chitin Stock Suspension (Modified from Hsu and Lockwood, 1975)

Finely divided commercial chitin (e.g., from Fluka or Sigma) is suspended in concentrated (32%) HCl. For 40 g of chitin, approximately 400 ml of HCl is required, but sometimes more HCl (600–800 ml) has to be used. Within about 30 min, a relatively thin, blackish collodial solution is obtained. The hydrochloric acid solution should never stand for more than 1 h at room temperature to avoid degradation of the chitin. The solution is poured into 2 liters of ice-cold water, upon which the chitin precipitates immediately as a pure white, fluffy material. It is collected on a separating funnel under suction and extensively washed. During these manipulations the material should never become dry, because drying makes it very difficult to resuspend. The washed precipitate is dialyzed against tap water for 12 to 24 h until the pH of the water remains above at least 4.5. Then enough distilled water is added to the chitin suspension to give a slurry which is sufficiently thin to be pipetted. The pH is adjusted to 7.2 with KOH. The exact volume is determined, and the approximate chitin content of the suspension calculated from the amount employed in the beginning. The material is distributed in convenient aliquots in bottles, autoclaved, and stored at 6°C. If the precipitated chitin appears too coarse, which may happen if the starting material was not well ground, it can be further homogenized with a blender.

Chitin agars are prepared best as overlay media, which saves chitin and gives clearer results, because the layer to be hydrolyzed is thinner and the material remains at the top of the plate, so that the bacteria have an easier access to it.

CT6 Agar

Top layer:

MgSO4 · 7H2O

0.1%

K2HPO4

0.02%

Agar

1.5%

Adjust the pH to 7.5. After autoclaving, enough of the sterile chitin suspension is added to give a good turbidity, which should be achieved with about 0.5% chitin and not more than 30% (by volume) of the suspension. The medium is poured as a thin layer on top of the following base agar:

Base agar:

Casitone (Difco)

0.1%

Yeast extract (Difco)

0.05%

MgSO4 · 7H2O

0.1%

Agar

1.2%

Adjust the pH to 7.2 and autoclave.

CT7 Agar

The top layer is as above; the base agar, however, is water (WAT) agar, which is as follows:

WAT agar:

CaCl2 · 2H2O

0.1%

Agar

1.5%

Adjust the pH to 7.2 and autoclave.

In CT7 agar, chitin is the only nitrogen, carbon, and energy source besides agar, and thus the medium is more selective. But not all chitin degraders grow on it. On CT6 agar, on the other hand, chitin degradation is sometimes suppressed by the peptone, and organisms that are not chitinovorous also grow on it.

Lysobacters decompose many bacteria, living and dead, and therefore may be isolated on such substrates. Strain AL-1 was obtained from lysis zones in lawns of Arthrobacter crystallopoietes (Ensign and Wolfe, 1965). Aqueous soil extracts were streaked on plates of medium A and incubated at 30°C.

Medium A

A basal agar with 0.5% peptone and 1% agar was over-laid with the same medium containing 109 cells/ml of Arthrobacter.

The bacteria from the plaques that developed in the lawn were purified by plating diluted suspensions on medium B.

Medium B

Agar, 1.5%, containing 109 cells/ml of washed Arthrobacter.

Presumably the Arthrobacter cells were living in the isolation medium and dead in the purification medium. Strain AL-1 was also maintained on medium B (at 30°C, with weekly transfers).

Similar isolation techniques may be devised with other bacteria and in fact have been used repeatedly to obtain lysobacters that lyse cyanobacteria. Stewart and Brown (1969) and Shilo (1970) isolated their organisms from water collected from a waste stabilization pond and a fish pond with a water bloom, respectively. Both groups used the soft-agar overlay technique developed for the isolation of cyanophages, and living Nostoc muscorum or Plectonema boryanum as the indicator organisms. Shilo (1970) first enriched the lytic agent by inoculating the sample into a Plectonema culture. While viral plaques appear after just 2–3 days, the bacterial plaques need 5–7 days to develop, and in addition are slightly sunken into the agar. Further, the bacteria also produce lysis zones in layers of autoclaved cyanobacteria. Shilo (1970) separated her strain from the cyanobacteria by transfer to a mineral salts liquid medium with 0.2% Casitone and incubation in the dark. The overlay technique may also be used for counting the lytic bacteria. Stewart and Brown (1969) determined for strain N-5 (myxobacter 44) a ratio of 1.3 for the number of viable cells to plaque-forming units.

A slightly different method was applied by Daft and Stewart (1971) and Daft et al. (1973). The cyanobacteria were grown on a suitable mineral salts medium with 0.6% agar. On the lawns produced after 7 days at 22–25°C, 1-ml water samples from the top-most layer of lakes or other surface waters, preferentially such with a cyanobacterial water bloom, were spread and the lysobacters isolated from the lysis zones that arose in the lawns. As indicator organisms, Nostoc ellipsosporum, Anabaena catenula, A. flos-aquae, or Phormidium foveolarum were used, and N. ellipsosporum (strain 1453/19 Cambridge University) proved particularly sensitive to lysis. The CP strains could be maintained without loss of lytic activity on CP agar.

CP Agar

K2HPO4

17 mg

MgCl2 · 6H2O

49 mg

CaCl2 · 6H2O

15 mg

NaCl

60 mg

FeCl3

0.3 mg

EDTA

7.4 mg

Standard trace element solution*

Casitone (Difco)

2 g

Agar

6 g

Distilled water

1 liter

The mineral part is that of ASM medium (Daft and Stewart, 1971) for the cultivation of algae (and probably dispensable for the lysobacters).

Footnote 1

Lysobacter colonies may be recognized by their spreading growth due to gliding motility, and colony morphology can thus be used as a lead to isolate the organisms. Thus, on plates designed for the isolation of myxobacteria and consisting of water agar (WAT agar) with streaks of living Escherichia coli that are inoculated with soil, we occasionally also observe swarming lysobacters.

The purification of Lysobacter strains is easy and is done either by plating of diluted cell suspensions or by making transfers from the advancing edge of swarm colonies. As swarming is much reduced or completely suppressed on rich media, the latter possibility exists only when the bacterium is grown on a lean medium like yeast agar or CY agar (see below). To avoid confluence of the arising colonies, plating is done best on a rich medium such as standard nutrient agar (Difco).

Cultivation

The lysobacters are robust, adaptable organisms, and their cultivation is no problem. They actually grow on any bacteriological standard medium. Christensen and Cook (1978) recommend the three following media:

PC Agar (Plate Count Agar, Difco)

Yeast extract

0.25%

Tryptone

0.5%

Glucose

0.1%

Agar

1.5%

CC Agar (Cook’s Cytophaga Agar; Christensen and Cook, 1972)

Tryptone

0.2%

Agar

1.0%

SA Agar (Skim-Milk Acetate Agar; Christensen and Cook, 1972)

Skim milk powder

0.5%

Yeast extract

0.05%

Sodium acetate

0.02%

Agar

1.5%

The pH for all of these media is adjusted to about 7.0 and the medium is sterilized by autoclaving. On PC agar, the colonies remain small and compact like those of nongliding bacteria, whereas on CC and SA agar, spreading swarms are obtained. Another good medium for lysobacters, including stock cultures, is the Yeast Agar mentioned above. On this medium the organisms form thin, spreading swarms, and because no or much less ammonia is produced than on the peptone media, the cultures tend to remain viable for longer time.

SA medium may also be used as a broth for liquid cultures, but a nonturbid medium is more convenient, e.g., 1% yeast extract medium (Ensign and Wolfe, 1966). We often use PEP medium.

PEP Medium

Peptone from casein, tryptically digested

1%

MgSO4 · 7H2O

0.1%

Adjust the pH to 7.2 and autoclave.

Other peptones can be used as well, e.g., Casitone (Difco), which is a pancreatic digest of casein, and casamino acids have also been employed successfully (Gillespie and Cook, 1965). L. brunescens appears not to grow well or to lyse early in pure peptone media. It could, however, be cultivated with a generation time of 4 h at 20–22°C in tryptone-starch medium.

Tryptone-Starch Medium (von Tigerstrom and Stelmaschuk, 1987b)

Tryptone

0.8%

Yeast extract

0.2%

Starch

0.3%

MgCl2

0.02%

A fully defined medium for L. enzymogenes strain AL-1 was developed by Ensign and is mentioned in Tan et al. (1974):

Defined Liquid Medium (for Strain AL-1)

Aspartic acid

0.2%

K2HPO4 · 3H2O

0.34% (0.03 M)

(NH4)2SO4

0.1%

MgSO4 · 7H2O

0.01%

Tap water

1%

Glucose

0.5%

Adjust to pH 7.0 and autoclave.

Although lysobacters tolerate relatively high pH values and can grow above pH 10 (Christensen and Cook, 1978), their optimum is between pH 7 and 9. While all appear to grow at 30°C, their temperature optimum is sometimes lower, and cultivation at 25°C or even 22°C may give better results. The organisms are strict aerobes, and liquid cultures have to be shaken.

The generation times are typically around 2.5 h (2.3 h for L. enzymogenes strain 495 in 0.8% yeast extract broth at 22°C: von Tigerstrom and Stelmaschuk, 1989; and 2.6 h for strain myxobacter 44 in a mineral salts medium with 0.2% starch and 0.25% peptone, 30°C: Stewart and Brown, 1971. Much longer generation times of 10.7 and 12.6 h are reported for growth under quasi-natural conditions in nutrient-poor culture filtrates from cyanobacteria and green algae, respectively (strain CP-1, 20°C: Fallowfield and Daft, 1988). A maximum cell yield of 2.5 g/l (dry weight) was obtained with strain 495 in condensed fish solubles with 2% glucose (Wah-On et al., 1980).

Large-scale fermentations with lysobacters have been performed for the production of the antibiotic myxin, and of an enzyme complex capable of lysing living yeast cells. While apparently nothing has been published about the industrial process developed by Hoffmann-La Roche (Nutley, NJ) for the manufacture of myxin with strain “Sorangium” 3C, some data on 10-liter fermentations are given in a Canadian patent (Can. Pat. 784,213; 30 April 1968). The fermentation was performed in the following medium: Tryptone, 0.1%; glucose, 0.1%; K2HPO4, 0.1%; MgSO4, 0.02%; CaCl2, 0.01%; and FeCl3, 0.001%; at pH 7.5 and 25°C. The aeration rate was 0.1 liter air per liter medium and min; the stirring rate, 300 rpm; the harvest time, 20 h with a 2% (v/v) inoculum, and 12 h with a 10% inoculum.

Production of yeast-lytic enzymes by “Cytophaga” NCIB 9497 was studied at up to a 900–1 liter scale (Asenjo et al., 1981). The medium consisted of 1% yeast extract and 1% glucose and permitted a maximum growth rate (µmax) of 0.36 h−1. The inoculum size was 3.8%, the initial pH 7.2, and the temperature 26°C. The aeration rate was set at 0.2 liter air per liter medium and min, and the impeller tip speed between 1.21 and 1.57 m·sec−1 (around 100 rpm). It appeared that a relatively low level of dissolved oxygen around 20–30% was favorable, particularly in the beginning. Polypropylene glycol 2000 was added as an antifoam agent and was well tolerated at 2 ml/1 but not higher. Still, after 24 h, the broth began to foam heavily. Harvest was at 30 h with a good yield of active enzyme, part of which was set free only by cell lysis at the end of the fermentation. Early cell lysis may be a serious problem. Obviously it is essential to start with young, vigorous seed cultures, e.g., from freeze-dried material.

Preservation

Stock cultures may be kept on yeast agar at room temperature (21°C) and should be transferred every 1 to 2 weeks. With stock cultures on peptone media, production of toxic levels of ammonia can be a problem. Storage of cultures in the cold is not recommended. In one reported case, all cells were dead after 3 weeks at 5°C (Asenjo et al., 1981). It seems understandable that the lytic enzymes produced by lysobacters also destabilize resting cultures.

Lysobacters survive freezing very well, either at −80°C or in liquid nitrogen. Cells from young plate cultures are suspended in PEP medium, or young shake cultures in the same medium are taken, and 1-ml samples are frozen without further precautions. The preserved cultures are reactivated by immersing them in tap water for a quick thawing and transferring the content as soon as it is liquefied. The longest storage period we have tested so far was 13 years at −80°C, which was reliably tolerated by all strains.

Lysobacters also can be lyophilized in skim milk by the standard procedure. It is reported that reactivated freeze-dried cultures may show a slightly different colony morphology and reduced bacteriolytic and proteolytic activity (Christensen, 1989). We dry the organisms with good results at room temperature, which may be less harmful to them. A few drops of a cell suspension in sterile skim milk are applied to a plug of lyophilized skim milk in an ampule. The wetted plug is then dried in vacuo, the ampule filled with nitrogen gas, and sealed.

Characterization

Morphologically, the various lysobacter species resemble one another closely (Christensen and Cook, 1978; Christensen, 1989). The cells are slender cylinders with rounded ends, and, at least in young healthy cultures, are rather regular in shape (Figs. 1 and 2). They typically measure 0.3–0.6 × 2–6 µm, but much longer threads, up to 70 µm, are usually also present. Those filaments obviously are incompletely divided cells and cell chains. A mixed population of short and long cells is very characteristic for the lysobacters and distinguishes them at once from any myxobacterium. To avert confusion, it should be mentioned that the morphological description given by Veldkamp (1955) of his chitinolytic strain does not fit the present NCIB 8501 strain, which indeed is a typical lysobacter. Instead, Veldkamp has given the morphology and cyclic-shape change of Flexibacter filiformis, which also is strongly chitinolytic.

Electron micrographs of thin sections show a typical Gram-negative cell wall, large mesosome-like structures in the center or at the poles, and granules interpreted as poly-β-hydroxybutyrate and polyphosphate. On the surface there may be ruthenium-red-positive material, presumably slime (Shilo, 1970; Stewart and Brown, 1971; Stewart and Daft, 1977); in other cases no such material was seen (“myxobacter” AL-1: Pate and Ordal, 1967). True rhapidosomes, i.e., the contracted tails of a defective phage (Reichenbach, 1967), were found in cell lysates of “Sorangium” 495 (Pate et al., 1967).

In contact with an interface, the lysobacters may move by gliding. The movements usually are rather slow but observable under the microscope. An average speed of 1 µm per min (30°C) has been reported (Veldkamp, 1955). When suspended in liquid the cells are nonmotile.

On lean media like CC, SA, and yeast agar, most lysobacters produce thin, filmlike swarms with little flamelike extensions at the edge (Figs. 3 and 4). Only L. gummosus appears not to spread on any medium, and its gummy colonies always show an entire edge. In general, the spreading of lysobacter swarms is not too fast, and it may take 2–3 weeks before they cover the entire plate. The swarm sheet itself may be somewhat slimy, but otherwise it is rather unstructured and smooth. This distinguishes the lysobacter swarms from myxobacterial swarms, which almost always are morphologically differentiated with radial veins, concentric ridges, and oscillating waves or ripples, and which in addition may etch themselves deeply into the agar surface (degenerate strains may produce homogeneous, slimy swarms). On rich media like PC agar, the colonies tend to remain compact, small, with a smooth convex surface and an entire edge. Those colonies often are highly mucoid. Enough slime may be produced to drop from the colony into the lid of the inverted dish (Ensign and Wolfe, 1965). As briefly mentioned, L. enzymogenes produces two different colonies, one dirty-white and mucoid, the other yellowish and nonmucoid. While both colony types are always obtained when starting from white colonies, the yellow ones yield only the yellow type. Physiologically and biochemically, the two organisms are identical (Christensen and Cook, 1978). Often, small, colorless crystals are seen within the colonies (Fig. 3e). In the case of L. gummosus, the colonies are of a rubbery consistency.

The color of colonies of Lysobacter may be off-white; cream; pale to deep yellow, sometimes with a greenish hue; pinkish; salmon; or orange-brown, depending on the species and on the medium. The chemical nature of those pigments is not known. The published absorption spectra of crude extracts show maxima or shoulders at 455, 482, and 516 nm (n-hexane) for salmon-colored strain FP-1; 448, 466, and 470 nm (ethanol) for three yellow “myxobacters” (Stewart and Brown, 1971); and 424, 442, and 464 nm (in methanol) for the yellow strains CP-1 and CP-15 (Daft and Stewart, 1971; Daft et al., 1973). While those spectra suggest a polyene chromophore, perhaps a carotenoid, they may be spectra of a mixture of pigments and thus cannot be reliably interpreted. Many strains produce a water-soluble, dark-brown, probably melanoid pigment, which is particularly prominent in old cultures and on media containing amino acids or peptones. Color reactions of phenolic compounds indicate the presence of a mono- and diphenol oxidase (Stewart and Brown, 1971). Deep-red crystals of myxin may appear within the colonies of L. antibioticus. Also, many cultures give off an unpleasant Pseudomonas- or pyridine-like odor.

In agitated liquid media, the lysobacters grow as homogeneous cell suspensions. When gently rotated, those cultures appear silky. Depending on the medium, liquid cultures may become somewhat viscous, and those of L. gummosus virtually solid.

Relatively little has been published about the composition of the lysobacter cell. A typical Gram-negative peptidoglycan was demonstrated in L. enzymogenes strain AL-1, with meso-diaminopimelic acid and D-alanine cross-bridges (Harcke et al., 1975). The inner and outer membrane could be separated starting from osmotically shocked cells. The outer membrane had a density of 1.30 g·cm−3 (CsCl), the inner one of 1.23 g·cm−3 (Hartmann et al., 1977). Ubiquinone Q-8 is the only respiratory quinone of L. antibioticus and L. enzymogenes (M. D. Collins, personal communication). This allows one to quickly distinguish lysobacters from myxobacteria as well as from organisms of the Cytophaga group (including high GC strains of Taxeobacter) which all contain menaquinones exclusively. The ribosomes of “myxobacter” 495 (L. enzymogenes) were difficult to isolate because tightly bound nucleases and proteases were attached to them and could not be removed by the usual methods (Sendecki et al., 1971). Otherwise, lysobacter ribosomes have the typical prokaryotic composition. The rRNA has the same base ratio as that of E. coli (GC/AU = 1.20). The presence of poly-β-hydroxybutyrate in the lytic “myxobacters” was substantiated by its conversion into crotonic acid (Stewart and Brown, 1971). The compound accumulates in the course of cultivation, with a maximum on the third day. Capnoids, a new type of sulfonolipids found in bacteria of the Cytophaga group (Godchaux and Leadbetter, 1983), and multicopy single-stranded DNA common in myxobacteria (Dhundale et al., 1985), are both absent in lysobacters. The lysobacter DNA has a high GC content of 65–71 mol% (Christensen and Cook, 1978; Daft and Stewart, 1971; Mitchell et al., 1969; Shilo, 1970; Stewart and Brown, 1971).

A number of metabolic enzymes have been studied in L. enzymogenes strain AL-1 (Guntermann et al., 1975; Hartmann et al., 1977). In the cytoplasm there is an α- and a β-glucosidase, a β-galactosidase, and an isocitrate dehydrogenase; on the cytoplasmic membrane, a succinate dehydrogenase; and bound to the outer membrane, an alkaline phosphatase and a N-acetyl glucosaminidase. The activities of those enzymes change during the cell cycle. Two distinct patterns were noted, one for the five hydrolases, the other for the two dehydrogenases. In the case of the α-glucosidase, the activity increase was shown to be due to de novo enzyme synthesis. While β-glucosidase and β-galactosidase are constitutive, α-glucosidase is inducible by maltose. In a study on the phylogenetic implications of the isozyme pattern of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (important for the synthesis of aromatic amino acids) in superfamily B (to which Lysobacter belongs according to 16S rRNA data), L. enzymogenes was found to possess only one enzyme variant, the one that is sensitive to feedback inhibition by tryptophan and, unique for Lysobacter, ultrasensitive to chorismate (Ahmad et al., 1986). This pattern is shared only by group V pseudomonads, which fits its classification based on 16S rRNA studies. In addition, the lysobacters produce a host of exoenzymes. As some of those are of a more general interest and have found applications, they will be discussed under “Practical Aspects,” this chapter.

For a large number of strains, comparative physiological and biochemical tests were performed and used for the differentiation of species (Christensen, 1989; Christensen and Cook, 1978). The strains studied for their ability to destroy cyanobacteria were not characterized in the same scheme as those able to destroy other bacteria, so that the following generalizations may not always apply to them.

The lysobacters are aerobic organisms, although many strains appear to grow best at a reduced oxygen level (10% O2). In the oxidation-fermentation (OF) test with glucose, practically all strains show oxidative growth but many also show fermentative growth. Growth of the lytic freshwater organisms stops immediately when the culture is flooded with N2 gas; no growth is obtained in paraffin-sealed tubes, and the generation time increases to 9 h (30°C) when the oxygen tension is reduced to 20% of the normal atmospheric pressure (Daft et al., 1975; Stewart and Brown, 1971). Catalase and oxidase are positive, but the catalase reaction is very sensitive to the culture conditions and sometimes is very weak or even absent (H. Reichenbach, unpublished observations).

The pH range is 4.5 to over 10, the optimum between 7 and 9. There is almost always a remarkable tolerance to alkaline conditions. Even the freshwater strains usually grow well at pH 9 (Stewart and Brown, 1971; Daft et al., 1975). The ability to grow at an acid pH varies with the different strains; many do not grow below pH 6. During growth on peptones, the pH may rise substantially due to ammonia production.

The temperature optimum is usually around 30°C, even for the freshwater organisms, but it varies substantially from isolate to isolate. It may be as low as 25°C or as high as 40°C. A few strains can grow at 2°C and at 50°C, but the limits often are 4 and 40°C.

The salt tolerance is usually limited to 1% NaCl or less, and no strain was found to grow in the presence of 3% NaCl.

The lysobacters are chemoorganotrophs. For many strains, NO3−, NH4+, glutamate, or asparagine serve as the sole nitrogen source whereas urea appears to be utilized only by a few strains. Some organisms, however, are more fastidious. Several of the strains that lyse cyanobacteria do not grow on inorganic nitrogen (Daft et al., 1973; Shilo, 1970), while others do (Stewart and Brown, 1971). All lysobacters grow on peptone as the only organic substrate, but the type and concentration of the peptone may be critical. Casitone (Difco) seems to be suitable in all cases, but strain FP-1 has a low optimum of 0.2% for Casitone and does not grow at 0.5%. The same strain also does not accept yeast extract, tryptone, or casamino acids, and does not grow on nutrient broth or nutrient agar (Shilo, 1970). In general, peptides appear to be the preferred carbon and energy source, and monosaccharides may only be used slowly (von Tigerstrom and Stelmaschuk, 1987b).

Glucose is probably utilized by all strains. Growth may take place on a wide spectrum of carbohydrates, e.g., in one case on: glycerol, mannitol, arabinose (weak), glucose, galactose, fructose (weak), mannose, lactose, sucrose, raffinose, and rhamnose (Veldkamp, 1955). Acid is produced from glucose and from several other sugars in a varying pattern that may be useful for the differentiation of species. Many strains grow on citrate as the sole carbon source.

All strains are strongly proteolytic as can be seen, e.g., by cultivation on skim milk agar. They liquefy gelatin, peptonize milk, and produce α-, β-, or γ-type hemolysis on sheep blood agar, including strain AL-1, which originally was reported to be negative for hemolysis (Ensign and Wolfe, 1965). Almost all lysobacters hydrolyze chitin. Veldkamp (1955) observed N-acetylglucosamine, glucosamine, acetic acid, and NH4+ as products. Several of the isolates that decompose cyanobacteria did not attack chitin (the failure was perhaps due to the method used; Stewart and Brown, 1969; Daft et al., 1975), while others do (Stewart and Brown, 1971). Crystalline cellulose (filter paper) is not decomposed, but many strains degrade carboxymethyl cellulose, and at least some strains can even grow on it as the only organic substrate (Stewart and Brown, 1971). While agar is not liquefied or softened, gelase fields (uncolored areas) may appear upon flooding with iodine solution (Shilo, 1970). Starch, pectate, and alginate may or may not be decomposed. Many strains produce lipases that cleave Tweens.

Ammonia is produced from proteins and peptones. Nitrate is only rarely reduced to nitrite. Some strains generate H2S. The indole, methyl red, and Voges-Proskauer tests are always negative, and the phosphatase test is always positive.

Sodium dodecyl sulfate usually diminishes growth at 0.01% and completely inhibits it at 0.1%. All lysobacters appear to be sensitive to polymyxin B, although the result may vary somewhat with the way the test is done (Christensen and Cook, 1978; Shilo, 1970; Stewart and Brown, 1971). This is a valuable distinguishing characteristic in so far as most Cytophaga-like bacteria are resistant to polymyxin B (Mitchell et al., 1969). All strains of L. brunescens as well as the freshwater lysobacters (Shilo, 1970; Stewart and Brown, 1971) also are rather sensitive to actinomycin D (around 2 µg/ml), which is in contrast to most Gram-negative bacteria but in accordance with the behavior of most other gliding bacteria (Dworkin, 1969). All L. brunescens strains are inhibited by chloramphenicol, penicillin G, and usually also streptomycin, but most other lysobacters are not. Strain FP-1 was also resistant to erythromycin and tetracycline, but inhibited by kanamycin and neomycin (Shilo, 1970). The strains of Stewart and Brown (1971) and of Daft et al. (1975) were sensitive to all these and several more antibiotics.

As already mentioned, the lysobacters are able to kill and disintegrate many living and healthy microorganisms, such as both Gram-positive and Gram-negative bacteria, including actinomycetes and cyanobacteria; filamentous fungi; algae; and even nematodes. Gram-negative strains are usually less readily killed and lysed than Gram-positive ones. The first signs of lysis may be observed within minutes after the culture broth has been added to a suspension of sensitive bacteria (Gillespie and Cook, 1965). The technique by which the lysobacters destroy cyanobacteria is particularly well documented (Daft and Stewart, 1971, 1973; Daft et al., 1973, 1975; Shilo, 1970; Stewart and Brown, 1970). They use two different mechanisms: 1) One strain, strain N-5 (myxobacter 44) attacks the cyanobacteria with free exoenzymes (Stewart and Brown, 1969, 1970, 1971). The first assault seems to be on the cell wall with lysozyme-like enzymes, but later the whole cell with the exception of the membranes is digested. Heterocysts and akinetes are more resistant but finally also destroyed. Only their walls remain. 2) All other strains need to be in contact with the cyanobacteria before they can lyse them (Daft and Stewart, 1973; Shilo, 1970). Since neither culture supernatants nor cell extracts are sufficient to disintegrate cyanobacteria on solid or in liquid media, enzymes bound to the surface of the lysobacter cell are probably involved. It appears that the lysobacters move actively toward their prey, perhaps attracted by the oxygen produced by photosynthesizing cells. They attach themselves with one cell pole often close to the cross-septa of the filaments, and then become fixed perpendicularly on the surface of the target cells. No special organelle for attachment is recognizable. The need for contact explains why lysis does not take place in shaken cultures. In unshaken liquid cultures, the lysobacters are found fixed to the cyanobacteria within 20 min after mixing. A single lysobacter cell may lyse a Nostoc cell within 20 min. It then moves on and attacks another cell in the filament. There is no specific order in which the cells along a filament are assaulted. When a lysobacter culture of a sufficiently high cell density is added to a cyanobacterial population, lysis may be complete within 1 to 3 h. The time required depends, of course, on environmental factors, notably oxygen and temperature. Thus, in a well-aerated culture at 26°C, Nostoc ellipsosporum is completely destroyed within 60 min. On ultrathin sections in the electron microscope, it can be seen that the first structure to disappear is the peptidoglycan layer. The cell contents are then gradually broken down, and only the cell membranes, lipid droplets adhering to them and, if present, the gas vacuoles remain. The latter rise to the surface where they can be collected. The membranes often coil up, looking in cross-sections like scrolls. Protoplasts may be released from the filaments. If there is an outer sheath, it also may disintegrate. In the case of Oscillatoria redekei, amorphous material disappears from the sheath and a fibrous layer is left. Heterocysts initially resist lysis but eventually also disintegrate, either under the impact of the bacterial enzymes or by autolytic processes started after the cells are released from the filaments. In infected cyanobacterial cultures, the chlorophyll a content and the nitrogenase activity go down concomitantly with lysis, so that either parameters can be used to measure its progress (Daft et al., 1973).

Tan et al. 1974 have devised a method to establish synchronized cultures of myxobacter AL-1: Cells of different sizes, equivalent to different stages of the growth cycle, were separated by centrifugation in a sucrose density gradient. As already mentioned, in this way, changes of enzyme activities during growth could be determined.

Taxonomy and Identification

The first step in the identification procedure is to make sure that an isolate belongs to the genus Lysobacter. An organism qualifies as a lysobacter if it is a chemoorganotrophic, aerobic, unicellular, gliding, Gram-negative bacterium with a GC content of 65 to 71 mol%. Those characteristics are, however, shared by the myxobacteria and by certain organisms of the Cytophaga group (such as Taxeobacter). Some myxobacteria have a very similar cell shape (for instance, Polyangium, Sorangium, and Chondromyces) but the populations never show the variation in cell length typical for lysobacters. The morphology and structure of myxobacterial swarms is usually completely different, myxobacteria produce fruiting bodies and myxospores, and they contain menaquinones instead of ubiquinones as respiratory quinones. The organisms of the Cytophaga group, as far as they are gliding, may form very similar soft, slimy swarm colonies, but their cells usually look much different, they often contain flexirubin-type pigments and always menaquinones. The colonies of most Cytophaga- like bacteria are more-or-less yellow to orange, often brightly colored, particularly on peptone agar, and turn red if covered with 20% KOH (flexirubin reaction), which is never observed with lysobacter strains. Unfortunately, Taxeobacter, the only genus that in its GC content comes close to Lysobacter, is red. It has a similar cell shape to Lysobacter, but its cells are much stouter and tend to arrange themselves side-by-side in a palisade like fashion. Also, Taxeobacter lacks the unpleasant smell of Lysobacter.

Presently, four Lysobacter species are recognized. Their differential characteristics are listed in Table 1. The separation of the species rests entirely on physiological and biochemical data and is based on phenotypic analysis of a large number of strains (Christensen and Cook, 1978). However, the relatively wide GC range of the strains of two species (65–70 mol% in L. enzymogenes and 66–69 mol% in L. antibioticus) and the substantial differences in the temperature maxima of strains of L. brunescens (37–50°C) suggest that more than one taxon may be hidden among the strains presently allocated to one species. First attempts to subdivide one of the species have been made (L. enzymogenes subsp. enzymogenes and subsp. cookii; Christensen and Cook, 1978), but before that can reasonably be done, the methods of molecular taxonomy, notably DNA-DNA hybridization, need to be used. It seems doubtful whether the occurrence of myxin can be used as a characteristic of the species L. antibioticus, because production of an antibiotic usually is strain- and not species-specific.

Table 1. Characteristics distinguishing the four species of Lysobacter.
Full size table

As there is a free exchange of organisms between soil and fresh water, the habitats given in Table 1 for the various species should not be taken as absolute. In fact, the “freshwater” lysobacters attacking cyanobacteria have also been found in soil (Daft et al., 1975), and we have isolated myxin-producing lysobacters from freshwater (H. Reichenbach, unpublished observations).

Two more species not shown in Table 1 also have been proposed L. lactamgenus (Ono et al., 1984) was studied because of its new cephem antibiotics, the cephabacins. While several characteristics of the strain are in accordance with a lysobacter, it differs substantially in its extremely high GC content (76 mol%, Tm) and in its inability to produce acid from any sugar. L. albus (Nozaki et al., 1987) synthesizes the novel antibiotic lactivicin. The description of this isolate is very preliminary and lacks essential data, e.g., on gliding motility and GC content. Acceptance of the new species will require that the strains be made freely accessible to others in the field.

Practical Aspects

The spectacular lytic capabilities of the lysobacters suggest that they may play an important role in the control of microbial populations in nature, although their population densities appear never to become high enough to cause a breakdown of a cyanobacterial water bloom (Daft et al., 1975). Still, it may be possible to eliminate blooming organisms in a limited area, or pathogenic or otherwise undesirable bacteria in sewage plants, like the filamentous organisms responsible for the bulking of sludge, by introducing lysobacter strains with strong, lytic enzymes. The potential is probably limited by the requirement for cell-to-cell contact as a prerequisite for lysis, which would restrict any application to nonagitated environments, such as trickling filters or dense, floating mats of the target organisms.

The ability of lysobacters to synthesize new secondary metabolites is of particular importance. The following antibiotics have been isolated from lysobacters (and Lysobacter-like organisms): the phenazine-N-oxide, myxin (Peterson et al., 1966; Weigele and Leimgruber, 1967); two cyclic decapeptides, myxosidin A and B (Clapin and Whitaker, 1976, 1978 Monahan and Whitaker, 1976); the quinoline compound, G 1499–2 (Evans et al., 1978); new cephem antibiotics, the cephabacins (Harada et al., 1984; Ono et al., 1984); the acyltetramic acids, catacandin A and B (Meyers et al., 1985); a dicyclic dipeptide with lactam-like properties, lactivicin (Harada et al., 1986, 1988 Nozaki et al., 1987, 1989); and a dibasic macrocyclic peptide lactone, lysobactin (Bonner et al., 1988; O’Sullivan et al., 1988; Tymiak et al., 1989).

Of those antibiotics, myxin has been produced industrially by Hoffmann-La Roche in Nutley for some time. It was applied in the form of a copper chelate called cuprimyxin and sold under the trade name Unitrop®. In contrast to free myxin, the chelate is very stable and practically insoluble. In contact with animal tissue, myxin is gradually released, so that the complex guarantees a sustained activity. Cuprimyxin was applied topically, mainly against skin infections including dermatophytes, and its use was restricted to veterinary medicine. The antibiotic is produced by strains of L. antibioticus, which was originally named “Sorangium” 3C (in the chemical literature, it is often further mislabelled as “Sporangium”). Myxin acts on DNA. There exists a voluminous literature on the various aspects of myxin, which can, however, not be reviewed here (see, e.g., on the chemistry: Sigg and Toth, 1967; Weigele and Leimgruber, 1967; Weigele et al., 1971; on the mechanism of action: Behki and Lesley, 1972; Lesley and Behki, 1967; on the biological activity and application: Grunberg et al., 1967; Maestrone and Brandt, 1980; Maestrone and Mitrovic, 1974; Maestrone et al., 1972; McDonald et al., 1980; Snyder and Imhoff, 1975).

The main reason why the lysobacters initially were noticed was their rich stock of exoenzymes. Several of those enzymes have a potential for an application in research or industry. One of the Lysobacter proteases has been studied in great detail for theoretical reasons and is one of the best-understood enzymes. Again, the literature on this topic is by far too extensive to be reviewed here comprehensively (see below).

Strain NCIB 8501 (if the present strain is really identical with the one first described, see above) was the first lysobacter to be studied for its exoenzymes, a chitinase complex (Veldkamp, 1955). The products of the enzymatic activity are N-acetylglucosamine, acetate, and ammonia, but not glucose. While chitin decomposition is a common feature of the lysobacters, it appears that so far no other study has been performed on the subject. An extracellular enzyme with both β-1,4-glucanase and chitosanase activity was purified from L. enzymogenes strain AL-1 (Hedges and Wolfe, 1974). It does not attack chitin. Enzymes of this type are probably responsible for the often-observed hydrolysis of carboxymethyl cellulose. They could also be useful for the study of the structure of fungal cell walls. Strain “Cytophaga” L1 (= NCIB 9497) was patented for its impressive combination of fast-working hydrolytic exoenzymes (Brit. Pat. 1,048,887, 23 November 1966). The enzyme complex includes chitinase, laminarinase, lipase, elastase, keratinase, and other proteolytic activity and may be useful for the decomposition of fungal mycelia from fermentations. Using the same strain, a process was later worked out for the degradation of living yeast cells (Andrews and Asenjo, 1984; Asenjo, 1980; Asenjo and Dunnill, 1981; Asenjo et al., 1981; Hunter and Asenjo, 1987a, 1987b). The yeast lytic enzymes are produced constitutively and consist of β-(1–6)-glucanase, mannanase, and high protease activities. The breakdown of the yeast cells is connected mainly with the protease and the glucanase activities, which have to act in sequence because a specific protease must remove the mannoprotein layer in the outer cell wall before the glucanase can become active. As the glucanase is repressed if more than 0.8 g of glucose is present per liter of medium, a 20-fold-higher enzyme yield is obtained in continuous cultures with optimal dilution rates compared with batch cultures. The lysobacter glucanase appears not to adsorb to the yeast glucan, attacks the fibrillar and the amorphous glucan fractions equally well, and shows no inhibition by its hydrolysis products. If crude enzyme preparations are applied in higher concentrations, the rate of lysis may decline. This may be due to competitive inhibition by carbohydrates present in the enzyme solution. The lysobacter enzyme mixture solubilizes the yeast cells almost completely and thus appears particularly useful for the production of yeast extract and of animal feed. A two-step model has been proposed to describe the kinetics of the process of lysis. Also, the separation of the enzyme complex from the culture broth and its application in an immobilized form has been studied. It appears, however, that the free enzyme is more promising for technical purposes.

Two alkaline phosphatases were demonstrated in L. enzymogenes strain 495 (von Tigerstrom, 1983; 1984; von Tigerstrom and Stelmaschuk, 1986, 1987a). The appearance of both activities is repressed by inorganic phosphate. One of those enzymes is excreted into the medium, has a molecular weight of about 25 kDa, and does not contain a metal ion, which is rather unusual for a bacterial phosphatase. As the enzyme is easy to recover and purify, it might be a useful tool in nucleic acid research. The other enzyme appears to be bound to the outer membrane, has a molecular weight of about 69 kDa, and seems to be composed of at least two subunits. It is a metal enzyme probably containing Zn2+, hydrolyzes a wide variety of 5′-,3′-, and 2′-ribose and -deoxyribose nucleotides, as well as sugar phosphates, and shows a remarkably high specific activity. A corresponding cell-bound phosphatase was also found in the three other Lysobacter species, and all four enzymes are immunologically related among themselves but completely different from cell-associated phosphatases from other gliding bacteria. The extracellular and the cell-bound phosphatase also differ in their amino acid composition.

The same strain produces two extracellular endonucleases (von Tigerstrom, 1980, 1981). One is a nonspecific nuclease (22–28 kDa) preferentially cleaving double-stranded DNA and, with reduced efficiency, single-stranded DNA and RNA, but not poly(A) and poly(C). It produces large oligonucleotides with 5′-phosphate groups. The synthesis of this enzyme is inhibited by RNA in the medium. The second enzyme is an RNAse (46–47 kDa) that also cleaves poly(A) and poly(C) but not double-and single-stranded DNA. It appears to have no base specificity. The enzyme is unusual in that it generates short oligonucleotides with 5′-phosphate ends (instead of the more common 3′-phosphate groups). The production of the RNAse is inhibited by phosphate in the medium. During its purification, a contaminating enzyme was discovered that degraded the dialysis bags. The extracellular enzymes of strain 495 are obtained with good yields in tryptone (0.4%) broth at 25°C (von Tigerstrom, 1983). While the production of nuclease, RNAse, and phosphatase is inhibited by Mg2+ and by Mn2+ above 0.1 and 0.01 mM, respectively, that of protease is stimulated by Mg2+ and neutral to Mn2+. The ions appear to interact specifically with the synthesis of the enzymes and not with their release from the cells.

Other hydrolases excreted by lysobacters are: endoamylases found in all four species (von Tigerstrom and Stelmaschuk, 1987b), which seems surprising because the characterization of the lysobacter species excludes starch hydrolysis in almost all strains of three species (see Table 1); the paradox may be a result of the test conditions. The enzyme of L. brunescens, the most potent organism, has a molecular weight of 47–49 kDa and also decomposes amylopectin, amylose, and glycogen, but not dextran. The enzyme probably has no commercial interest because of its enzymatic properties and low yield. Further, all species produce two extracellular lipolytic esterases (von Tigerstrom and Stelmaschuk, 1989). One is excreted into the medium and is inducible by olive oil. As it is synthesized only after exponential growth, it appears to be under catabolite repression. The other enzyme is associated with the outer membrane and is constitutive. The two esterases differ in their substrate specificity. Both are very active on Tween 20, but only the free enzyme is able to attack olive oil. In addition, there is a third, cytoplasmic esterase which, in contrast to the extracellular enzymes, is very active on tributyrin. Finally, there is a periplasmic β-lactamase.

The most fascinating Lysobacter enzymes are their proteases. Two proteases were isolated from the culture supernatant of L. enzymogenes strain (“Sorangium” or “myxobacter”) 495; one of them α-lytic protease, became a very important enzyme for scientific reasons. The strain was originally noted for its ability to rapidly lyse a wide spectrum of bacteria (Gillespie and Cook, 1965) and nematodes (Katznelson et al., 1964). Soon it was realized that the lytic activity was due to at least two different proteases, α- and β-lytic proteases (Whitaker, 1965), and procedures were worked out to produce and purify those enzymes (Whitaker et al., 1965a; Whitaker, 1967a). Later it was found that there are at least two more proteases in the culture broth.

A major research effort has been made to characterize the α-lytic protease. This enzyme is an alkaline serine protease (for a summary of the early work see: Whitaker, 1970) that aroused great interest because it was the first bacterial serine protease with the same amino acid sequence in its active center, Gly-Asp-Ser-Gly-Gly, as the mammalian pancreatic serine proteases (Olson et al., 1970; Whitaker et al., 1966; Whitaker and Roy, 1967), and not Thr-Ser-Met as in most other bacterial serine proteases. This suggested a common ancestor for both enzymes and invited studies on the evolution of protein structure and function. The similarity to porcine pancreatic elastase is particularly notable, a similarity which also extends to substrate specificity and kinetic properties (Kaplan and Whitaker, 1969; Kaplan et al., 1970). While the homology in the amino acid sequence is only high in certain critical regions (overall homology 18%), long sections appear conserved in the three-dimensional structure as revealed by high-resolution X-ray analyses and molecular models (55% topological equivalence). There is an even higher correspondence with Streptomyces griseus proteases A and B, with 35% and 36% sequence homology and 80% topological equivalence (e.g., Brayer et al., 1979; Fujinaga et al., 1985; McLachlan and Shotton, 1971). The α-lytic protease (EC 3.4.21.12; 19.8 kDa) consists of 198 amino acids and contains three disulfide bridges. It cleaves polypeptides at the carbonyl side of short neutral aliphatic amino acids, preferentially alanine (Kaplan and Whitaker, 1969). It also attacks bacterial peptidoglycans, mainly at the cross-linkages (Tsai et al., 1965). Like other serine proteases, α-lytic protease has several subsites (in this case, six) for substrate binding, with different amino acid specificities (Bauer et al., 1981). This explains why the enzyme prefers long substrate molecules over short ones. While the pH optimum for catalytic activity is reached at pH 8, substrate binding is independent of pH between pH 5 and 10.5 (Kaplan and Whitaker, 1969; Paterson and Whitaker, 1969). The enzyme shows a high stability unparalleled in any other protease (Kaplan et al., 1970), which appears to arise from a high structural rigidity (Fujinaga et al., 1985). As with other serine proteases, a strong H-isotope effect is observed: the catalytic rate drops to one-third in 2H2O (Hunkapiller et al., 1973). The discovery that α-lytic protease contains but one histidine (His) residue (Juráek and Whitaker, 1967) proved particularly momentous for the elucidation of the catalytic mechanism of serine proteases, for the other enzymes all contain more than one His. This finding at once excluded a requirement of several His residues for the catalytic process, as was postulated before. The histidine in α-lytic protease is homologous with the His-57 in α-chymotrypsin and is part of the catalytic triad, Asp-102/His-57/Ser-195 (the adopted numbering is that of α-chymotrypsin). By the use of a His auxotrophic mutant, 13C and 15N could substantially be enriched in His-57 (Westler et al., 1982), which allowed refined NMR studies of the dissociation behavior and hydrogen bonding in the active center (Bachovkin, 1986; Bachovkin et al., 1981; Hunkapiller et al., 1973). Another approach was the use of peptide-boronic acid inhibitors forming rather stable enzyme-substrate complexes, which can be analyzed as models of the otherwise-not-accessible transition state (Bone et al., 1987; 1989). Those studies provided deep insights into the mechanism of catalysis and substrate specificity. The gene of α-lytic protease (the progene) has also been cloned in Escherichia coli (Epstein and Wensink, 1988; Silen et al., 1988). At the amino end of the protease domain, the gene has an open reading frame that extends 198 (or 199: the two articles do not completely agree in the details) amino acid codons upstream and represents a very large prepro sequence. The latter consists of a typical bacterial pre (or signal) peptide of 24 (or 33) amino acids ending with a Ala-Leu-Ala-Ala cleavage site, followed by a pro region of 174 (or 166) amino acids, i.e., more than twice the size of other known bacterial propeptides. The high sequence homology observed between α-lytic protease and proteases A and B of Streptomyces griseus continues for 49 amino acids into the pro region (21% and 38% homology, respectively), and then falls suddenly to a low value of 7% and 3%. This suggests that the Lysobacter pro region is a composite of two parts, perhaps of different origin and with different functions. In fact the amino acid sequence around the cleavage site of the signal peptide shows a unique homology to a trypsin inhibitor of maize, and it was speculated that this part of the peptide may block the protease activity, while the conservative part may serve an identical function as the (much shorter) propeptides of proteinases A and B, namely, arranging the correct folding of the protease domain (Epstein and Wensink, 1988). The Lysobacter ribosomal binding site (AGGAG) is homologous to known E. coli binding sites. When the Lysobacter gene is fused with an inducible E. coli promoter and signal sequence (e.g., pho A), α-lytic protease is expressed in E. coli and (probably unspecifically) exported into the medium (Silen and Agard, 1989; Silen et al., 1989). Deletion of the pro region results in inactive enzyme, which is immunologically and electrophoretically indistinguishable from α-lytic protease, but presumably improperly folded. Site-specific mutation of the Ser-195 in the catalytic pocket leads to the production of a double-sized inactive protease molecule, which suggests that the removal of the propeptide is an autocatalytic process. Surprisingly, the activation (not the production) of α-lytic protease proved temperature sensitive and did not take place above 30°C. If the pro region and the protease domain were cloned separately with independently inducible E. coli promoters, active enzyme was obtained when both were induced concomitantly, in spite of the fact that in this case, the two peptide were not covalently connected. Obviously, the propeptide functions as a chaperonin-like, though specific, folding template. It appears that α-lytic protease is produced exclusively by strain 495.

Much less is known about the β-lytic protease, a second protease from strain 495. The enzyme is a nonserine protease and resembles mammalian carboxypeptidase and particularly the bacterial neutral proteases (Oza, 1973). Its specificity is quite distinct from that of the pancreatopeptidases. It cleaves very selectively at the carbonyl side of neutral amino acids. Thus, only the B chain of (denatured) insulin is cleaved, and that between glycine-23 and phenylalanine-24 and, much more slowly, between valine-18 and cysteine-19 (Whitaker et al., 1965b). A free α-amino group or a free terminal carboxyl next to the cleavage site is not accepted, but an amido group on the carboxyl already eliminates the blocking effect of the latter. The β-enzyme is more active than the α-enzyme on bacterial peptidoglycan rapidly hydrolyzing the muramic-acid alanine bond (Tsai et al., 1965). The β-lytic protease (19 kDa) appears to be composed of 177 amino acids and to contain two disulfide bridges and one atom of zinc (Juráek and Whitaker, 1967). The zinc can be removed and restored again with reconstitution of enzyme activity (Whitaker, 1967b). While the apoenzyme is no longer proteolytic (Oza, 1973), the cell-wall-lytic activity was still observed (Whitaker and Roy, 1967). But conceivably the protease was simply restored by a contamination of the peptidoglycan with zinc. The pH optimum of β-lytic protease is 6.5 (Oza, 1973). The proteases of strain 495 can be produced with good yields (4–4.5 g of α- and 2 g of β-enzyme from 100 liters) on casamino acid/glucose media (Whitaker, 1967a). Also, production on media with condensed fish solubles, a by-product of the salmon canneries, seems feasible (Wah-On et al., 1980).

Finally, the proteases of L. enzymogenes strain (“myxobacter”) AL-1 (ATCC 27796) should briefly be mentioned. The organism was isolated because it lysed cells and cell walls of Arthrobacter crystallopoietes, and enzymes with that property were of much interest at that time for studies on the cell wall structure of bacteria (Ensign and Wolfe, 1965, 1966). In fact, one of the AL-1 enzymes was soon successfully applied for that purpose (for a review, see Ghuysen, 1968). The enzyme is particularly useful because it cleaves the bond between muramic acid and L-alanine, making it possible to isolate the pure polysaccharide backbones of peptidoglycan. The enzyme could thus be used to demonstrate a shortening of the polysaccharide chain length during the rod-to-sphere conversion of Arthrobacter (Krulwich et al., 1967). At first, only the enzyme just mentioned, AL-1 protease or, later, AL-1 protease I, was recognized and characterized (Ensign and Wolfe, 1966; Jackson and Wolfe, 1968; Jackson and Matsueda, 1970). The enzyme can be produced by fermentation in 1% yeast extract medium at 30°C for 36 h, and is recovered from the culture supernatant with a yield of 350 mg of pure enzyme from 100 liters. The AL-1 protease I (13.5 kDa) is an alkaline endopeptidase with a sharp pH optimum at 9.0. The molecule consists of 136 amino acids, with Ser, Asp, Gly, Thr, and Ala accounting for more than 50% of the residues. It contains one disulfide bridge, and does not contain a hexose molecule, as was previously suggested. The enzyme is spherical, compact, and very stable. Within minutes, it completely lyses whole cells of many Gram-positive and some Gram-negative bacteria, as well as purified peptidoglycan (Ensign and Wolfe, 1965). It cleaves about 30% of the bonds in casein and albumin, and 15% in gelatin. In the B chain of insulin only the bonds between Ala and Leu, Gly and Phe, and Val and Cys are attacked. Thus it appears that a hydrophobic residue is required at the cleavage site, but the exact specificity is not yet known. In peptidoglycan, besides the connection of the peptide side chain to the polysaccharide backbone, only the bonds between terminal D-Ala and the pentaglycine or L-Ala bridges, and bonds within the pentaglycine bridge are cleaved. The enzyme therefore became a useful tool for the elucidation of the structure of bacterial cell walls (e.g., Ghuysen, 1968; Jarvis and Strominger, 1967; Katz and Strominger, 1967; Tipper, 1969).

Also, the second protease from strain AL-1, AL-1 protease II (17 kDa), is a small, stable, alkaline endopeptidase, but it does not attack bacterial cell walls (Wingard et al., 1972). It is obtained from the culture supernatant with a yield of 10% of protease I. The enzyme is very unusual in two respects: it readily crystallizes from the column fractions, and it cleaves exclusively at the amino side of lysine. It does not remove terminal lysine residues. The enzyme consists of 157 amino acids, 50% of which are Asp, Ala, Thr, and Gly, and has a broad pH optimum between 8.5 and 9.0.

A third unusual protease from AL-1 is called endoproteinase Lys-C. This enzyme has a high although not absolute specificity for peptide and ester bonds at the carboxyl end of lysine (information leaflet Boehringer Mannheim, Biochemica-Dienst no. 42, April 1980), so that it can be used for sequencing proteins and for peptide mapping (Au et al., 1989; Boileau et al., 1982; Hofsteenge et al., 1983; Jekel et al., 1983; Jolles et al., 1983). The enzyme (30 kDa unreduced, 33 kDa reduced) is an alkaline serine protease, is very stable even in the presence of 0.1% sodium dodecylsulfate or 5 M urea, and is commercially available from Boehringer Mannheim (Mannheim, Germany).