secondary metabolites of the fluorescent pseudomonads · fluorescent pseudomonad secondary...

MICROBIOLOGICAL REVIEWS, Sept. 1979, p. 422442 Vol. 4:3, No. :30146-0749/79/03-0422/21$02.00/0

Secondary Metabolites of the Fluorescent PseudomonadsT. LEISINGER* AND R. MARGRAFFt

Mikrobiologisches Institut, Eidgenossische Technische Hochschule, ETH-Zentrum, CH-8092 Zurich,Switzerland

INTRODUCTION ............................................................. 422PA¶TERN OF SECONDARY METABOLITES PRODUCED BY PSEUDOMO-NADS 422

LIPIDS AND RELATED COMPOUNDS 427PSEUDOMONIC ACID ........................ .... 429PHENAZINES 429PYRROLNITRIN ............ ... .............. .. .. 430PEPTllDES AND AMINO ACIDS

.................................. 432FLUORESCENT PIGMENTS 434MANIPULATION OF SECONDARY METABOLITE PRODUCTION IN PSEU-DOMONAS ........................ ..... .... 435

SUMMARY AND CONCLUSIONS ........................... .... .. 436LITERATURE CMD ........................... 437

INTRODUCTIONMicrobiologists working in the field of second-

ary metabolism are confronted with the ques-tion: What is the role of secondary metabolitesin the physiology and ecology of the producingorganisms? An answer to this problem wouldnot only enlarge our understanding of an impor-tant biological phenomenon, but it might alsoprovide industrial microbiologists with strate-gies for increasing the yield of economically im-portant microbial products. In recent years ithas become obvious that there is no generalsolution to this enigma, but rather there are asmany answers as there are secondary metabo-lites (153). It has also become apparent that anunderstanding of the controls affecting the pro-duction of a particular secondary metabolite canonly be reached by extensive studies on its bio-synthetic pathway, on the regulatory mecha-nisms involved in its formation, and on the ge-netics and physiology of the producing organism(63). It is obvious to choose secondary metabo-lites of industrial importance and strains with ahigh producing capacity for studies on secondarymetabolism. In the past this approach has beenfollowed by industrial microbiologists, and,while leading to considerable practical successin increasing the yields of antibiotics, it hasfailed to reveal the regulatory processes govern-ing the production of secondary metabolites.There are numerous reports offering phenome-nological descriptions of the parameters affect-ing the formation of secondary metabolites, butthere is a definite lack of studies at the biochem-ical and genetic levels. In part this may be due

t Permanent address: Rhone-Poulenc Industries, Centre deRecherches Nicolas Grillet, F-94400 Vitry s/Seine, France.

to the complex biosynthetic pathways of manysecondary metabolites, to the variety of orga-nisms studied, to the absence of suitable geneticsystems, and to the association of the formationof many secondary metabolites with processesof differentiation, adding a further dimension ofcomplexity to the regulatory mechanisms in-volved.

In the present contribution we summarize in-formation on the production of secondary me-tabolites by pseudomonads, with special empha-sis on the fluorescent pseudomonads. The moreimportant groups of secondary metabolites arediscussed and, where available, information ontheir biosynthesis and their mode of action isreviewed. Pseudomonads represent the majorgroup of non-differentiating microorganismsproducing antibiotics. The lack of cytologicaland physiological differentiation makes it possi-ble to study the effect of various factors onsecondary metabolism without interferencefrom genetically programmed development cy-cles. The pseudomonads may therefore offerexperimental advantages over the filamentousfungi, the actinomycetes, and the bacilli forstudying certain facets of secondary metabolism.

PATTERN OF SECONDARYMETABOLITES LITES PRODUCED BY

PSEUDOMONADSFluorescent pseudomonads, the most inten-

sively studied group among Pseudomonas spe-cies, comprise P. aeruginosa, the type species ofthe genus, P. fluorescens (four biotypes), P.putida (two biotypes), P. chlororaphis, P. au-reofaciens, and the two plant pathogenic speciesP. cichorii and P. syringae. The latter speciesincludes a large number of nomenspecies (23).

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FLUORESCENT PSEUDOMONAD SECONDARY METABOLITES 423

All fluorescent pseudomonads fall into one ofthe five "ribonucleic acid (RNA) homologygroups" defined by ribosomal RNA-deoxyribo-nucleic acid competition experiments (109). Theguanine-plus-cytosine content of their deoxyri-bonucleic acid ranges from 58 to 68 mol% (108).In common with the other species of the genusPseudomonas, the fluorescent pseudomonadsare gram-negative, strictly aerobic, polarly flag-ellated rods. The group is quite heterogeneousand, apart from the ability to produce water-soluble fluorescent pigments of unknown chem-ical structure, there are no phenotypic propertiescommon to all members of the group. In recentyears the genetics and biochemistry ofpseudom-onads, especially of P. aeruginosa and P. putida,have been explored intensively (20, 59). Thewide interest in these organisms stems from therole of P. aeruginosa as an opportunistic path-ogen in humans, from the enormous catabolicpotential of the pseudomonads, from their usein the industrial conversion of organic chemicals,and from the plant diseases caused by somemembers of the genus.Apart from the pigments, most secondary me-

tabolites produced by the fluorescent pseudom-onads have been detected and investigated be-cause of their antibiotic activity. The practicaluse of antibiotics from pseudomonads dates backto the period before the "antibiotic era." In 1899Emmerich and L6w (29) reported that the cell-free culture fluid of P. aeruginosa, concentratedto one-tenth of its original volume, killed severalkinds of bacteria. Because the preparation ex-hibited enzymatic activities, it was called pyo-cyanase. It has been used extensively in thetherapy of diphtheria, influenza, and meningitisduring the first two decades of this century.Pyocyanase has been produced commercially,but its clinical use was abandoned as the prep-arations, probably because of strain degenera-tion, became less potent and varied in efficacy.During the antibiotic era approximately 50 dif-ferent antibiotic substances from pseudomonadshave been discovered (5). Only two of these,pyocyanine and pyrrolnitrin, have been pro-duced on a commercial basis. Although thisnumber may increase in the future, it is clearthat the significance of the pseudomonads asproducers of industrially important secondarymetabolites is very limited.The known chemical structures of secondary

metabolites produced by fluorescent pseudom-onads are listed in Table 1. Most of them exhibitantibiotic or phytotoxic activity. These second-ary metabolites were detected as a result of theirbiological activity and, as is the case with othermicroorganisms, secondary products devoid of

antibiotic activity have escaped detection. Table1, therefore, probably represents a distorted pic-ture of the secondary metabolites from pseu-domonads. Nevertheless, it can be said that com-pared to the variety of antibiotics produced bythe Actinomycetales and the fungi, antibioticsproduced by the pseudomonads cover a morerestricted range of chemical structures. Macro-lides, aminoglycosides, polyenes, quinone-typeantibiotics, oxygen-containing heterocycles, andalicyclic antibiotics have not been found amongthe secondary metabolites produced by thepseudomonads. Most antibiotics isolated fromPseudomonas culture filtrates, namely, phena-zines, pyrrolnitrin-type antibiotics, pyo com-pounds, and indole derivatives, fall into the classof N-containing heterocycles and have beenshown to originate from intermediates or endproducts of the aromatic amino acid biosyn-thetic pathway. Another substantial class ofPseudomonas secondary products comprisesunusual amino acids and peptides. In addition tothese two major groups of secondary metabo-lites, some glycolipids, lipids, and aliphatic com-pounds have been isolated from Pseudomonascultures and are listed in Table 1.

In a few cases it is possible to correlate thepattern of secondary metabolites of pseudomo-nads with metabolic or ecological properties spe-cific for this group of organisms. Although thepresence of most antibiotics from various orga-nisms cannot be demonstrated in the naturalenvironment of the producing organisms (40), ithas been demonstrated that some phytotoxinsare produced by pseudomonads in infectedplants, the natural habitat of the phytopatho-genic pseudomonads (102). It thus seems thatproduction of phytotoxins is one of the rareexamples where the production of secondarymetabolites confers obvious selective advantagesto the producing organisms, enabling them, to-gether with other, as yet unknown factors, tocolonize specific host plants, i.e., ecologicalniches otherwise not accessible (111). Slime pro-duction by P. aeruginosa furnishes another ex-ample of a selective advantage due to the for-mation of a secondary metabolite. From respi-ratory infections, accompanying cystic fibrosis,mucoid mutants of P. aeruginosa can be isolated(31, 41). The production of an alginate-type het-eropolysaccharide (8), consisting of,-1,4-linkedD-mannuronic acid and L-guluronic acid andcontaining O-acetyl groups (31), leads to in-creased resistance to carbenicillin and to phageresistance of such strains. Since spontaneouslyarising nonmucoid revertants have a growth rateadvantage, mucoid strains are unstable underlaboratory conditions (41, 96), whereas the eco-

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424 LEISINGER AND MARGRAFF

TABLE 1. Secondary metabolites from fluorescent pseudomonads

Compound (trivial name) Structure Reference

LIPIDS

Pyo compounds (pseudanes)

2-n Pentyl-4-quinolinolPyo lb = pseudane VIIPyo Ic = pseudane IXA'-Pseudane VIIPyo III = A'-pseudane IX2-Alkyl-4-hydroxyquinoline N-oxides2-(2-Heptenyl)-3-methyl-4-quinolinol

Rhamnolipids

Pyolipic acidCompound B

Jarvis rhamnolipidCompound A

PHENAZINES

PyocyanineHemipyocanineIdoininPhenazine-l-carboxylic acid (tubermycin B)ChlororaphinOxychlororaphinAeruginosin AAeruginosin B

PYRROLES

Pyoluteorin

OH

Rl'2NIR

R, RN=(CH2)4CH H-(CH2)6CH3 -H-(CH2)sCH3 -H-CH=CH(CH2)4CH:, -H-CH=CH(CH2),;CH:i -H-(CH2),CH fi, n = 6, 8, 10 (N-oxide) -H-CH2CH=CH(CH2):,CH:l -CH,HO - -CHCH2COOCHCH2COOH

(CH2)6 (CH2)6

IIOH C3 CH3

RR-H-COCH=CH(CH2)6CH:,HO O-CHCH2COOCHCH2COOH

VHNI3H(CH2)6 (C,H2)6

OH O CH3 CH3HO\

OH 0

RR-H-COCH=CH(CH2)6CH:,

9 t IRg NJ R2

R6 4Rr,

R,-OH-OH-OH-COOH-CONH2-CONH2-H-H

R2-H-H-H-H-H-H-NH2-NH2

Rr,=CH3

-o

-H

R,;-H-H-H-H-H-H-COOH-{COOH

R-=H-H-H-H-H-H-H-SO3H

QtcoaOH -1N Cl

H

150535381532149

6152

26, 57, 72, 73152

Rio,

-O

-H

-CH3-H:,

3737373737375854

136

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TABLE 1-ContinuedCompound (trivial name)

Phenylpyrroles

PyrrolnitrinIsopyrrolnitrin2-ChloropyrrolnitrinAminopyrrolnitrinOxypyrrolnitrinMonodechloropyrrolnitrin4-(2'-Amino-3'-chlorophenyl)pyrrole-2-car-

boxylic acid

INDOLES

3-ChloroindoleIndole-3-carboxaldehyde6-Bromoindole-3-carboxaldehyde7-Chloroindoleacetic acidIndoleacryloisonitrile

AMINO ACIDS and PEPTIDES

L-2-Amino-4-methoxy trans-3-butenoic acid

O-Ethyl homoserine

Phaseotoxin A

Structure

R: HR 4

5 H

R2-H-Cl-H-H-H-H-COOH

R3-Cl-Cl-Cl-Cl-Cl-CH-H

4

R6)9~N) 2

R7 H

-cl-CHO-CHO--HO-CH2COOH-CH=CH-No-C

R2'-NO,-NO2-NO2-NH2-NO2-NO2-NH2

R=-H-H-Br-H-H

R,-H-H-H-Cl-H

HCH300- C-CCH-COOH

HHNH2CH3CHiO-CH2-CH2 CH-COOH

NH2

0If

HO - P - NH-CH-COOHOH CH2-CH -COOH

Phaseolotoxins

Phaseolotoxin(2-Serine)-phaseolotoxin

Tabtoxins

Tabtoxin(2-Serine)-tabtoxin

Isotabtoxins

Isotabtoxin(2-Serine)-isotabtoxin

Tabtoxinine

H2N-CH-CO-NH-CH-CO-NH-CH- COOH

(CH2)3 CH2R (CH2)4NH - OSO-NH2 NH-C-NH2

OH NHR-H

OHHOCO-CH -NH-CO -CH -CH2- CH2f-I

CHOH NH2 NF0R

R

-H

OH,2CH2- NH2

HOCO-CH-NH-CO NbOH

CHOH

R-H3-H

OHHOCO - CH - CH2-CH2- C - COOH

NH2 C;H2- NH2

Reference

R--Cl-H-Cl-C1-C1-H-C1

Ri-H-H-H-H-OH-H-H

674745394850126

8515015012531

124, 128

105

114

100102

131, 140131, 140

131, 140131, 140

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426 LEISINGER AND MARGRA

Compound (trivial name)

Tabtoxinine-8-lactam

Tabtoxinine-8-lactam

Coronatine

Proferroroanmine A

Pyrimine

Viscosin

PTERINES

Pterine6-Aminopterine6-HydroxymethylpterineMonapterine

Ribityllumazine6-Methyl-ribityllumazinePutidolumazine

6-(p-Hydroxyphenyl)-rnbityllumazine

6-(3-Indolyl)-ribityllumazine

MISCELLANEOUS COMPOUNDSCyanhydric acid

TABLE 1-ContinuedStructure

OHHOCO-CH -CHC-Ci

NH2 j-NH

OH

JI?tCH2-NH2HOCO N

H

0

/CONH,.

HOCO>L

Q-yOCCOH

IVHP

CO-CH7-CH-CH-COOHNH,

D-13-hydroxydecanoyl-L-Leu-D-Glu--

-D -atlo -Thr- D-VaI -L- Leu - D - Ser

tL1-le+D - Ser L - Leut-

N*4NyR

R-H-NH2-CH20H-CHOHCHOHCH20H

NNtNz

cHEHOIocH2oN

R-H-CH3-CH2CH2COOH

-Q}OH

H

HCN

MICROBIOL. REV.

Reference

25

140

66

115, 116

129

56

142142142135, 141, 143

135134134

134

134

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TABLE 1-Continued

Compound (trivial name) Structure Reference

Aeruginoic acid

Magnesidin

Pseudomonic acid

Pseudomonic acid A

Pseudomonic acid B

Antibiotic P-2563

P-2563 (A)P-2563 (P)

Amino-2-acetophenone

C-acetyl phloroglucinols

OH xCOOH

0 >OOCO(CHH2)6CH3

CH3CH^k>O 1/2MCHH nR4,

OH( H3 HOt -'CO~CO(CHA)COOH

H3C W ~ R CH3OH R

R-H

-OHCH20H

R-HN

HO40

H2NCH2CH-CH-CH-CH-CH2OHOH H2N OH

R-COCH3-COCH2CH3

COCH3

NH2

OH

HOT OH

R2=COCH,-COCH3-COCH:,

Antibotic DB-2073 (alkylresorcinol)

R4-H-COCH:,-COCH:,

Rb-H-H-COCH,

OH

CH3-(CH2 /(CHAC5-CH3OH

" A.-G. Hoechst, British patent 1,478,643, July 1977 (Chem. Abstr. 88:168480x, 1978).^ Yoshitomi Pharmaceutical Industries Ltd., Japan Kokai 7770,083, June 1977 (Chem Abstr. 87:182603a, 1977).

logical conditions in the infected tissue appar-

ently favor the selection of slime-producing mu-tants.

LIPIDS AND RELATED COMPOUNDSIn 1945, while reinvestigating the antibiotic

substances produced by P. aeruginosa, Hays etal. (53) obtained active preparations upon ex-tracting sedimented cells with ethanol. From theethanol extract they separated a number of an-

tibiotic substances which were named pyo com-pounds. Four of the five substances obtained,namely, pyo Ib, Ic, II, and III, were shown to bestructurally related. The structures of pyo Ib, Ic,and III were determined and confirmed by syn-thesis (145, 146). All pyo compounds are moreactive against gram-positive than against gram-negative bacteria.A group of substances closely related to the

pyo compounds was detected in the course of

151

Hoechst'

4, 16, Yoshi-tomib

17

103103

89

9, 1209, 34, 1209, 120

74, 76

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studies aimed at identifying a streptomycin an-tagonist excreted by P. aeruginosa (133). Thefactor responsible for this antagonism was iso-lated (83) and shown to consist of a mixture of2-n heptyl-, 2-n nonyl-, and 2-n undecyl-4-hy-droxyquinoline N-oxides (21). These com-pounds, which probably correspond to pyo II,antagonized the inhibition by streptomycin anddihydrostreptomycin of Bacillus subtilis andStaphylococcus aureus but not of gram-negativebacteria. They were able to antagonize the ac-tion of 1,000 times their weight of dihydrostrep-tomycin and at higher concentrations inhibitedgrowth of gram-positive bacteria. Although themode ofaction of the 2-alkyl-4-hydroxyquinolineN-oxides in antagonizing the inhibitory action ofdihydrostreptomycin has not been elucidated, itwas observed that the compounds inhibited elec-tron transport by cytochromes. This effect wasassumed to be the reason for their inhibitoryaction on bacterial growth (84). Pyo compoundshave been rediscovered by Russian workers (80,81), who named them pseudanes, and more re-cently they were also detected in a marine pseu-domonad (150).The isolation of anthranilic acid and 2-n hep-

tyl-3-oxy-4-quinolene from a "pyo" fermentationbroth (137, 138) strongly suggested that the pyocompounds are condensation products betweenanthranilic acid and a fatty acid precursor ac-cording to the scheme proposed by Cornforthand James (21), as depicted in Fig. 1. Thisscheme was corroborated by following the incor-poration into 2-n heptyl-hydroxyquinoline of['4COOH]anthranilic acid, ['5N]anthranilic acid,[14CH3]acetate, and ['4COOH]malonic acid fedto P. aeruginosa (122). The distribution of ra-dioactivity in the product was determined aftercleaving the molecule by ozonolysis into an-thranilic acid, a fatty acid, and carbon dioxidefrom the C3 of the quinoline ring. The entiremolecule of anthranilic acid was incorporatedinto 2-n heptyl-hydroxyquinoline, and the f3-keto decanoic acid moiety of the product wasshown to originate from four malonate units andone acetate unit according to the pattern shownin Fig. 2.

In addition to its possible role as a precursorof the pyo compounds, decanoic acid forms partof a number of other secondary metabolites frompseudomonads. D-,8-Hydroxy-n-decanoic acid isa component of pyolipic acid, of the rhamnolip-ids, of the cyclodepsipeptide viscosin, and pre-sumably of magnesidin (Table 1). This fatty acidhas also been identified as a constituent of thelipopolysaccharide layer of the outer membraneof certain gram-negative bacteria, includingpseudomonads (121).Bergstrom et al. (6) reported the extraction of

COOHN NH2

ICO(CH2

+ IoC o

0 R

0OH

N N RH

DH 0OOOH

6N

OH

N R

FIG. 1. Biosynthesis of pyo compounds from an-thranilic acid and a /3-keto acid intermediate asproposed by Cornforth and James (21).

+ 0

CH3 COOHo aCH2(COOH)2

.,

0

A a .

0 0 3H

FIG. 2. Incorporation of '4C-labeled acetate andmalonate into pyo Ib (pseudane VII) according toRitter (122).

pyolipic acid, an antibiotic active against Myco-bacterium tuberculosis, from cells of P. aerugi-nosa. By titration, an average molecular weightof 500 was determined for pyolipic acid. Uponacid hydrolysis the substance yielded 27% rham-nose and 73% of a fatty acid fraction consistingmainly of f-hydroxydecanoic acid, with ,B-hy-droxyoctanoic and f3-hydroxydodecanic acidrepresenting minor components. Pyolipic acidwas thought to be identical to L-rhamnosyl-,f-hydroxydecanoic acid, which has a molecularweight of 334 (98). However, the stoichiometryof the components found in the acid hydrolysateof the compound corresponds to L-rhamnosyl-,f-hydroxydecanoyl-1-decanoic acid, which hasa molecular weight of 502 and contains 74% ,8-hydroxydecanoic acid.A similar glycolipid was isolated by Jarvis and

Johnson (73) from the culture fluid of P. aerugi-nosa grown on glycerol. It was first identified asL-rhamnosyl-(1,3)-L-rhamnosyl-,8-hydroxydeca-noyl-,8-hydroxydecanoic acid. Later it was estab-lished that the correct linkage between the tworhamnose moieties was not 1,3 but 1,2 (26). Thecompound is excreted into the medium duringthe stationary phase of growth, and the optimalconditions for its formation from various carbonsources have been defined (51, 52). Subsequentlythe enzymatic synthesis of this rhamnolipid insonic extracts of P. aeruginosa has been studied.

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Burger et al. (10) showed that its synthesis pro-

ceeds by the following three reactions:

2 fl-hydroxydecanoyl-CoA + H20

Bf-hydroxydecanoyl-,8-hydroxydecanoate (1)

+ 2 CoA

TDP-L-rhamnose

+ ,3-hydroxydecanoyl-,8-hydroxydecanoateTDP + L-rhamnosyl-f3-hydroxydecanoyl-

f3-hydroxydecanoate

TDP-L-rhamnose + L-rhamnosyl-

B8-hydroxydecanoyl-,8-hydroxydecanoate (3)

-- TDP + L-rhamnosyl-L-rhamnosyl-

,8-hydroxydecanoyl-f8-hydroxydecanoatewhere CoA is coenzyme A and TDP is thymidinediphosphate.Although the exact mechanism of formation

of 83-hydroxydecanoyl-,8-hydroxydecanoate (re-action 1) remains unclear, it has been shownthat the synthesis of the rhamnolipid involvestwo sequential glycosyl transfer reactions (reac-tions 2 and 3), each catalyzed by a specific rham-nosyl transferase. The latter two enzymes were

separated from each other and partially purified(10). L -Rhamnosyl -,8 -hydroxydecanoyl -,8 - hy-droxydecanoate, which is identical to pyolipicacid, was isolated from culture broths of P.aeruginosa grown on n-paraffins (71, 72). It wasbactericidal for gram-positive bacteria. Further-more, both mono- and dirhamnolipids exhibitedmycoplasmacidal and antiviral activities in vitro.Because of their emulsifying activities the rham-nolipids stimulated growth of P. aeruginosa on

n-paraffms (57, 72). In a recent study a pseudo-monad isolated from oil-soaked soil was shownto produce a new type of rhamnolipid duringgrowth on n-paraffins. In addition to one or twomolecules of rhamnose and two molecules of ,B-hydroxydecanoic acid, the compounds also con-tained a-decenoic acid. They exhibited strongemulsifying activity in an oil-water system, andtheir use in activated sludge treatment of hydro-carbon-polluted water is under investigation(152).

PSEUDOMONIC ACIDPseudomonic acid A (Table 1) was isolated in

1971 from a strain of P. fluorescens (4). It isresponsible for a significant proportion of theantibacterial activity in the culture fluid of thisorganism. Its structure (16, 17) and its absolute

stereochemistry (1) have been determined. Thecompound is mainly active against gram-positiveorganisms, whereas gram-negative bacteria, withthe exception of Neisseria and Haemophilusiare relatively insensitive to pseudomonic acid.At low concentrations the antibiotic is bacterio-static; at higher concentrations it is bactericidal(R. Sutherland, K. R. Comber, L. W. Mizen, B.Slocombe, and J. P. Clayton, Program Abstr.Intersci. Conf. Antimicrob. Agents Chemother.16th, Chicago, Ill., Abstr. no. 52, 1976). Its lowminimum inhibitory concentration for Neisseriagonorrhoeae (0.01 ,ug/ml) may lead to its prac-tical use against penicillin-resistant Neisseriastrains (J. P. Clayton [Beecham Group Ltd.],German patent 2,746,974, April 1978 [Chem.Abstr. 89:36919h, 1978].Pseudomonic acid showed no cross-resistance

with a wide range of antibiotics, and its uniquestructure seems to be correlated with a novelmode of action (64). Protein synthesis in S.aureus was shown to be inhibited by pseu-domonic acid. The antibiotic also inhibited RNAsynthesis, an effect which was prevented by theaddition of chloramphenicol to the test orga-nism. This latter observation suggested thatpseudomonic acid led to starvation for one orseveral amino acids or to the inhibition of theaminoacylation of transfer RNA, thereby caus-ing the arrest of RNA synthesis in organismswith stringent control ofRNA formation. Chlor-amphenicol and some other antibiotics acting atthe ribosomal level are known to abolish strin-gent RNA control. The antagonistic effect ofchloramphenicol on the inhibition of RNA syn-thesis by pseudomonic acid might thus be ex-plained by the current views on the regulationof RNA synthesis.

PHENAZINESThe production of phenazine pigments by a

variety of procaryotes such as Streptomyces,Streptosporangium, Microbispora, Brevibac-terium, Sorangium, and Pseudomonas has beenreported. Microorganisms constitute the exclu-sive source of phenazines in nature, and there isa considerable overlap in the production pattern:several different organisms produce the samecompound and a particular microorganism mayoften produce a variety of phenazines (11, 12,75). Approximately 30 different phenazine com-pounds have been described so far (37). Phena-zines are isolated by extraction of culture fil-trates with organic solvents. Among pseudomo-nads they are produced by some members of theP. cepacia group and by many strains belongingto the fluorescent pseudomonads. Therefore,most studies on the production and biosynthetic

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pathways of the various phenazine pigmentshave been conducted with representatives of thefluorescent pseudomonads, in particular with P.aeruginosa, which produces the blue pigmentpyocyanine, and with P. aureofaciens, whichexcretes primarily phenazine-1-carboxylic acidinto the medium.The involvement of the aromatic amino acid

pathway in the biosynthesis of pyocyanine wasfirst suggested by the incorporation of ['4C]shi-kimic acid and ['4C]quinic acid into pyocyanine(87, 99). These early experiments were impededby the redistribution of the labeled precursorsthrough degradative pathways. In the subse-quent studies on pyocyanine biosynthesis var-ious mutants of P. aeruginosa have proven use-ful. Using a mutant blocked in the catabolism ofshikimic acid and quinic acid, Ingledew andCampbell (68) were able to show that all carbonatoms of the phenazine nucleus of pyocyaninewere derived from shikimic acid. Quinic acid,anthranilic acid, tryptophan, tyrosine, and phen-ylalanine did not serve as precursor molecules.By analyzing mutants blocked in different stepsof aromatic amino acid biosynthesis (13, 86)chorismate was identified as the branch pointmetabolite leading off the aromatic pathway topyocyanine. Genetic blocks before shikimate didnot affect the formation of pyocyanine on shi-kimate-supplemented medium. Blocks betweenshikimate and chorismate abolished pyocyanineproduction on media containing the requiredaromatic amino acids and shikimate, whereaslesions in the enzymes of the aromatic aminoacid biosyntheses after chorismic acid were with-out effect on pyocyanine formation.The pathway from chorismate to the individ-

ual phenazines is still obscure. Hollstein andMcCamey (61) concluded from incorporation ex-periments with specifically labeled D-["C]shi-kimic acid that a common precursor of the phen-azines arises by the symmetrical condensation oftwo identically substituted molecules of chor-ismic acid. Although a technique for the recog-nition of pyocyanine mutants of P. aeruginosahas been devised (14), the most recent studieson phenazine biosynthesis using pigmentationmutants have been conducted with P. phenazin-ium, a nonfluorescent pseudomonad ofuncertaintaxonomical status (11, 12). Two properties ofthis organism make it a suitable object for study-ing phenazine biosynthesis: it produces ten dif-ferent phenazines and, on a minimal mediumcontaining L-threonine as the only source ofcarbon and nitrogen, it incorporates, 25% of thecarbon of threonine into phenazines. Under sim-ilar growth conditions it incorporates about 70%of the carbon of D-['4C]shikimate, mainly into

iodinin. Cross-feeding experiments with pigmen-tation mutants (11), as well as feeding experi-ments in which radioactive phenazines isolatedfrom culture media were added to growing cul-tures of the wild type and of pigmentation mu-tants, led Byng and Turner (12) to propose ascheme for phenazine biosynthesis, which is rep-resented in Fig. 3. In this hypothetical pathwayphenazine-1,6-dicarboxylate is the common pre-cursor of the phenazines produced by P. phen-azinium. From this branch point compound oneroute would lead via two consecutive hydrox-ylative decarboxylations and two N-oxidationsto 1,6-dihydroxyphenazine 5,10-dioxide (iodi-nin). Alternatively, phenazine-1,6-dicarboxylatewould be decarboxylated to yield phenazine-1-carboxylate, which in turn is viewed as the pre-cursor of the phenazines metabolized to 1,8-di-hydroxyphenazine 10-monoxide (Fig. 3). Directevidence for a pathway with phenazine-1,6-di-carboxylate as a key branch point compoundhas been obtained by the observations that thedimethyl ester of "C-labeled phenazine-1,6-di-carboxylate is metabolized by P. phenaziniumin significant amounts to iodinin (A. M. Messen-ger and J. M. Turner, personal communication)and by P. aureofaciens to phenazine-1-carbox-ylate (60). Incorporation experiments in whichthe free acid of phenazine-1,6-dicarboxylate wasused instead of its dimethyl ester were unsuc-cessful, presumably due to permeability prob-lems. The findings of Holliman and collaborators(32, 46, 55) on the formation of phenazines by P.aureofaciens and P. aeruginosa may be accom-modated in the pathway of Fig. 3 by assumingphenazine-l-carboxylate as the second majorbranch point. This key intermediate would actas the precursor of pyocyanine, 1-hydroxyphen-azine and its 10-monoxide, aeruginosins A andB, and 2-hydroxyphenazine and its 1-carboxyl-ate.

PYRROLNITRINPyrrolnitrin, an antifungal antibiotic produced

by many fluorescent and nonfluorescent strainsof the genus Pseudomonas, was first describedby Arima et al. (2, 3). Gorman and Lively (39)have reviewed early information on the biosyn-thetic pathway and the fermentative productionof pyrrolnitrin. The antibiotic is primarily usedagainst dermatophytic fungi, especially againstmembers of the genus Trichophyton. Isopyrrol-nitrin, oxypyrrolnitrin, and monodechloropyr-rolnitrin have lower antifungal activities thanpyrrolnitrin. Elander et al. (27) surveyed 29Pseudomonas strains classified as P. chlorora-phis, P. aureofaciens, and P. cepacia for theproduction of pyrrolnitrin. Four strains of P.

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1 -Hydroxy-phenazine10- monoxide2)

t1- Hydroxy-phenazine

OH

Chorismic acid

+ COOH9 10 1

8 \2

/ 3N /

COOH 5 4

Phenazine-1,6-dicarboxylate

6- Hydroxyphenazine-l - carboxylate

1,6 - Dihydroxyphenazine

2 -Hydroxy-phenazine3)

2 - Hydroxy -phenazine-l-carboxybate

2-Amino- 5-methyl- 1-Hydroxy-5-methyl-phenazine - 6 - phenazinecarboxylate (Pyocyanine)"

W (Aeruginosin A)")

COOH

/ \5-Methylphenazine-l-carboxylate

N

Phenazine -1-carboxylate

9- Hydroxyphenazine-I -carboxylate

2,9 - DihYdroxyphenazine -1-carboxylate

1,6 -Dihydroxyphenazine -5,10 -dioxide(lodinin)'

1,8- Dihydroxyphenazine

1,8 - Dihydroxyphenazine- 10-monoxide°

FIG. 3. Hypothetical scheme forphenazine biosynthesis in Pseudomonas species. Modified after Byng andTurner (12). Incorporation of radioactive precursors according to this scheme has been observed for the endproducts formed by: 1) P. phenazinium (12), 2) P. iodina (Brevibacterium iodinum) (55), 3) P. aureofaciens(32), 4) P. aeruginosa (32, 46).

cepacia and four strains belonging to the twofluorescent species excreted pyrrolnitrin into themedium. A maximum level of about 100 ,ug/mlwas reached by a strain of P. cepacia.Whereas it is well established that pyrrolnitrin

is formed in several steps from tryptophan, thedetails of this pathway have not been elucidated.Studies with P. aureofaciens have shown thattryptophan is a precursor of pyrrolnitrin. Addi-tion of D-tryptophan to the medium resulted ina marked increase in pyrrolnitrin formation. L-Tryptophan had no effect in this respect, butboth labeled enantiomers were incorporated intothe product (85). Martin et al. (91), feedingtryptophan isotopically labeled in various posi-tions to P. aureofaciens, demonstrated that theamino nitrogen of D-tryptophan gave rise to thenitro group of pyrrolnitrin. The C2 of the indolenucleus was retained and the C3 of the side

chain became C3 of the pyrrole. Tritium at thechiral carbon of the amino acid was retainedduring biosynthesis when L-tryptophan was fed,but not in the case of D-tryptophan. These re-sults supported pathway A (39) shown in Fig. 4.More recently, Floss et al. (33), using trypto-

phan labeled with '3C in the 3-position of thechain and with '5N in the amino group, showedthat L- rather than D-tryptophan was the im-mediate precursor of pyrrolnitrin, despite thefact that under any condition tested, "4C-labeledD-tryptophan was incorporated more efficientlyinto pyrrolnitrin than the L-isomer. The sameauthors prepared and fed to P. aureofaciens anumber of presumed precursors of pyrrolnitrin.(Amino-2-phenyl-)-3-pyrrole (Fig. 4) was effi-ciently incorporated into pyrrolnitrin. This ledthem to propose a modified pathway (pathwayB in Fig. 4) for pyrrolnitrin formation. According

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to this proposal chlorine must be introduced intothe fl-position of the pyrrole ring, although elec-trophilic substitution of pyrroles normally oc-curs in the a-position.The order of introduction of the two chlorine

atoms in pyrrolnitrin biosynthesis remainedlargely speculative until recently, when a newmetabolite was isolated at an early stage of afermentation with P. aureofaciens by Salcher etal. (126). (Amino-2-chloro-3-phenyl-)-4-pyrrole-2-carboxylic acid (Table 1), after chlorinationand subsequent decarboxylation, yields amino-

pyrrolnitrin, a direct precursor of pyrrolnitrin.The same authors also isolated 7-chloroindole-3-acetic acid and 3-chloroanthranilic acid fromthe fermentation broth of the same organism(125). According to these results 7-chlorotryp-tophan is viewed as a common precursor of 3-chloroanthranilic acid, 7-chloroindole-3-aceticacid, and pyrrolnitrin (pathway C in Fig. 4).

PEPTIDES AND AMINO ACIDSThe phytopathogenic pseudomonads belong-

ing to the species P. syringae produce low-mo-

H.COOH

H

A

ClQJyO(COOH

CL

N N Nl COOHH H

O HCOOH

N N2

CL H

2hu7N COOHH H

KwkNH N-COOHHH

Ci H

I ~~~~~~~~ICl H

FIG. 4. Three different pathways for the conversion of tryptophan to pyrrolnitrin suggested by (A) Gormanand Lively (39), (B) Floss et al. (33), and (C) Salcher et al. (126).

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lecular-weight phytotoxins of a peptide or aminoacid nature. The role of these toxins in plantdisease, their chemistry, and their mode of ac-

tion have been reviewed by Patil (110) and byStrobel (132). The structure of tabtoxin (Table1), one of the toxins produced by P. tabaci, thecausal agent of wildfire disease of tobacco, hasbeen established by Stewart (131). It is a dipep-tide consisting of tabtoxinine-fl-lactam and L-

threonine. All strains forming tabtoxin also pro-

duce (2-serine)-tabtoxin (140). Tabtoxin and (2-serine)-tabtoxin isomerize easily to the biologi-cally inactive isotabtoxins by intramoleculartranslactamization. Tabtoxinine is formed byhydrolysis of tabtoxin or isotabtoxin. In additionto tabtoxin and (2-serine)-tabtoxin, the 8- andf)-lactams of tabtoxinine have been detected inculture filtrates of P. tabaci (25, 140). Tabtoxin,(2-serine)-tabtoxin, and tabtoxinine-/1-lactamhave been shown to cause chlorosis in tissues ofvarious plants (25). The biosynthesis of the tox-ins from P. tabaci is unknown, and their modeof action in the plant remains to be determined.

P. phaseolicola is responsible for the diseaseknown as halo blight of beans. It produces a

number of extracellular phytotoxins which havebeen studied extensively in recent years. Therole of the halo blight toxins in disease is firmlyestablished. Culture filtrates of P. phaseolicolaor purified toxin preparations cause the chloroticsymptoms found on naturally infected plants.Furthermore, the accumulation of ornithine typ-ical for chlorotic plant tissue correlated with thespecific inhibition of plant ornithine transcar-bamylase in vitro by purified toxin(s) (112, 113,139). The chlorosis in bean leaves was reversedby the addition of citrulline and arginine, whichis consistent with the cause of chlorosis beingdue to an inhibition of omithine transcarbamyl-ase. Recently, the toxins from culture filtrates ofP. phaseolicola have been purified and chemi-cally characterized by two research groups. Patilet al. (114) demonstrated the presence of at leastfour ornithine transcarbamylase-inhibiting com-

pounds separable on diethylaminoethyl-Sepha-dex. One component, phaseotoxin A, was iden-tified as N-phosphoglutamic acid, whereas thestructures of the other three components are

still unknown. The production of N-phospho-glutamic acid by P. phaseolicola is the first caseof a naturally occurring N-phosphorylated pri-mary amine. The unusual structure of phaseo-toxin A is strongly supported by the fact thatchemically synthesized N-phosphoglutamic acidinhibited ornithine transcarbamylase and in-duced chlorosis in bean leaves (114). Mitchell(100), on the other hand, isolated and character-ized the tripeptide (N5-phosphosulfamyl)-orni-

thyl-alanyl-homoarginine as the toxin responsi-ble for bean halo blight. This major compoundwas given the trivial name phaseolotoxin. (2-Serine)-phaseolotoxin was produced as a minorcomponent by the same organism (102). Phas-eolotoxin, (N5-phosphosulfamyl)-ornithyl-alanine, and (N5-phosphosulfamyl)-ornithinecaused chlorosis and ornithine accumulation inbean leaves (100). Since phaseolotoxin is rapidlydegraded to (N5-phosphosulfamyl)-ornithine inplant tissue, Mitchell proposed that the lattercompound is primarily responsible for the actualtoxic effect on plants (101). The effects of nutri-tional components, amino acid supplements, andtemperature on toxin production by P. phaseo-licola in liquid culture have recently been inves-tigated (123). Wide variations in toxin yield havebeen observed, and it was suggested that theconflicting results regarding the chemical com-position of the toxin from this organism mightbe due to the production of different toxinsdepending on the composition of the culturemedium.

Phytotoxins from other phytopathogenic no-menspecies of P. syringae are not well charac-terized. The toxins of P. coronafaciens, thepathogen of oats, are related to the tabtoxins,whereas the toxins of P. glycinea and P. tomato,the pathogens of soybean and tomato, seem tohave chromatographic and biological propertiessimilar to those of the bean halo blight toxins(110, 132). P. syringae, the pathogen of maizeand wheat, produces syringomycin, a wide-spec-trum antibiotic that is also phytotoxic (22). Theantibiotic has been partially purified and shownto be a small peptide yielding nine differentamino acids upon hydrolysis (130). More re-cently, homogeneous syringomycin preparationshave been obtained by thin-layer chromatogra-phy and polyacrylamide gel electrophoresis, butthe structure of the phytotoxic antibiotic is stillunknown (43). It was observed that the acridineorange-induced elimination of a 22 x 106-daltonplasmid from P. syringae was correlated withthe loss of the ability to produce syringomycin(C. F. Gonzales and A. K. Vidaver, Proc. Am.Phytopathol. Soc. 4:107, 1977). There exists alsopreliminary evidence for plasmid involvement inthe synthesis of phaseotoxin and tabtoxin (62).

L-2-Amino-4-methoxy-trans-3-butenoic acid(AMB), an amino acid antimetabolite inhibitingthe growth of gram-positive and gram-negativebacteria on miniimal media, is produced by P.aeruginosa (124, 128). AMB was formed by dif-ferent strains of P. aeruginosa on complex andminimal media containing either glucose or n-paraffins as sources of carbon and energy. Themaximum levels of antimetabolite produced

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were 18 mg/liter. Its minimal inhibitory concen-tration for Escherichia coli on minimal mediumwas as low as 3 x 10' M. Interestingly, thereversal patterns of the growth inhibition causedby AMB were different for different indicatorbacteria. In B. subtilis growth inhibition wasrelieved by D- and L-alanine, D- and L-2-amino-butanoic acid, D-glutamic acid, and D-asparticacid (128), whereas methionine, homocysteine,and cystathionine reversed growth inhibition inE. coli (124). A metabolic explanation for theinhibition caused by AMB is still lacking. How-ever, the toxin has been shown to act as anirreversible inhibitor of pyridoxal-linked aspar-tate-aminotransferase (118, 119).The enol ether group of AMB is rare among

naturally occurring amino acids and has beenobserved only in two structurally related anti-metabolites: rhizobitoxine [2-amino-4-(2-amino-3-hydroxypropoxy)-trans-3-butenoic acid] fromRhizobium japonicum (107) and L-2-amino-4-(2-aminoethoxy)-trans-3-butenoic acid from aStreptomyces species (117). In the culture brothsof both organisms the saturated inactive com-pounds dihydrorhizobitoxine (106) and L-2-amino-4-(2-aminoethoxy)butanoic acid (127)were found. Addition of synthetic D,L-2-amino-4-(2-aminoethoxy)butanoic acid to the Strepto-myces culture medium led to a threefold-en-hanced production of the corresponding unsat-urated antimetabolite, suggesting that the satu-rated amino acid serves as a precursor for theantimetabolite (127).Although the biosynthesis of AMB is not

known, preliminary studies have shown that theaddition of L-homoserine or synthetic 0-methyl-D,L-homoserine to the culture medium of P.aeruginosa strain PA01 resulted in a threefoldincrease in AMB production. This observationand the fact that the label from L-[`CH3]methi-onine was incorporated with 50% efficiency intothe methoxy group of AMB suggest that AMBis formed from homoserine by O-methylationand subsequent dehydrogenation (unpublisheddata).

FLUORESCENT PIGMENTSUnder certain growth conditions all repre-

sentatives of the fluorescent pseudomonads pro-duce water-soluble, yellow-green, fluorescentpigments which have been called bacterial fluo-rescein, fluorescein, or pyoverdine. The term"pyoverdine" is preferable to the designations"bacterial fluorescein" or "fluorescein," whichare likely to be confused with the chemicallysynthesized fluorescein (resorcinolphthalein).The pyoverdines formed by different species offluorescent pseudomonads may be distinguished

by a suffix indicating the producing species (94).The excitation maximum of the pyoverdines isaround 400 nm, with an emission maximnum ofaround 500 nm. Due to the difficulties encoun-tered during the isolation and purification of thefragile molecules, their chemical structures re-main largely unknown, and several hypothesesas to the nature of the fluorescent chromophorehave been formulated. Lenhoff (82) and Greppinand Gouda (42) have postulated a pyrrole deriv-ative by analogy with the cytochromes. A ribo-flavin component of bacterial fluorescein wassuspected by Birkofer and Birkofer (7), and apteridine chromophore was suggested by Giral(38) and Naves (R. G. Naves, Ph.D. thesis, Uni-versity of Geneva, Geneva, Switzerland, 1955).Chakrabarty and Roy (19) isolated a compoundfrom cultures of P. fluorescens with a molecularweight of only 210 which had physiochemicalproperties similar to those of the pteridines. Inthese early studies the molecular weights of thefluorescent pigments were presumably underes-timated, and the fluorescent compounds isolatedmay have been degradation products of pyo-verdines or other fluorescent metabolites pro-duced by pseudomonads.More recent studies have revealed that pyo-

verdines have molecular weights in the range of1,000. In partially purified preparations, ob-tained by gel filtration, the fluorescent pigmentswere found to consist of a peptide associatedwith a fluorescent chromophore. The peptidefrom P. mildenbergii contained serine, threo-nine, glutamic acid, and lysine (65, 104), whereasthe peptide in the fluorescent pigment of P.fluorescens (Migula) consisted of serine, glycine,glutamic acid, ornithine, and lysine (97). A sig-nificant contribution to the understanding of thechemistry, biosynthesis, and physiological func-tion of pyoverdinepf, the fluorescent pigment ofP. fluorescens, was made by Meyer and Abdal-lah (94). They observed that pyoverdinepfformed an extremely stable ferric complex andcould be purified in this form by a methoddeveloped for the purification of siderochromes(154). P. fluorescens produced only one molec-ular species of pyoverdine with a molecularweight of 1,500, which at slightly alkaline pHwas transformed into a number of degradationproducts. Obviously these derivatives have pre-viously been attributed to different pigment spe-cies. Although the chemical structure of pyo-verdinepf is not fully established, it probablyconsists of a quinoline chromophore associatedwith a cyclic peptide (no free amino group) anda short aliphatic chain (J. M. Meyer, Thesed'Etat, University Louis Pasteur, Strasbourg,France, 1977). Upon hydrolysis of the peptide

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moiety, serine, glycine, alanine, and N5-hy-droxyornithine were detected. The unusualamino acid N5-hydroxyornithine is a constituentof many siderochromes, including the sidero-chrome polypeptide ferribatin isolated from cul-tures of P. fluorescens (Migula) (92). In fact, thefollowing evidence has been presented (95) thatpyoverdinepf is a typical iron chelator with aspecific role in the transport of Fe3" into P.fluorescens. (i) Formation of pyoverdinepf oc-curred only in iron-deficient bacteria and wasnot influenced by other nutritional factors, theoxygenation rate, the pH, or by illumination.Under iron-limited growth in medium contain-ing 135 Ig of Fe3+ per liter the amount ofpigmentproduced was almost equal to the cellular dryweight. (ii) Pyoverdinepf formed a very stableferric complex with a stability constant of thesame order as those of other siderochromes. Ithad a low affinity for ferrous ions. (iii) The rateof iron uptake by P. fluorescens increased whenpurified pyoverdinepf was added to washed cells.Iron transport was an active process.The physiological function demonstrated for

pyoverdinepf has also been described for fluores-cent iron transport hydroxamates of unknownchemical structure produced by an unidentifiedfluorescent pseudomonad (35). The biosynthesisof iron transport compounds by this organismwas temperature sensitive: at 20°C 100 ug ofFe3" per liter led to maximal cell yield, whereasat 31°C growth depended on the addition of3,000 ,ug of Fe3+ per liter, as well as on supple-mentation of the medium with hydroxamateiron compounds produced by the organism atlower temperatures (35). It will be interesting tosee whether a possible common role of the flu-orescent pigments from different pseudomonadsin iron transport is paralleled by identical orsimilar chemical structures.

Fluorescent metabolites different from pyo-verdines include the ribityllumazines isolatedfrom cultures of P. putida, putidolumazine, apossible intermediate in riboflavine biosynthesis(134, 135), 6-hydroxymethylpterine, a precursorof folic acid found in cultures of P. roseus fluo-rescens (142), and D-erythroneopterine formedby P. putida (135) (Table 1).

MANIPULATION OF SECONDARYMETABOLITE PRODUCTION IN

PSEUDOMONASLittle effort has been directed towards increas-

ing the secondary metabolite production of pseu-domonads by mutation or by systematic opti-mization of medium composition and growthconditions. The limited practical use of productsfrom pseudomonads has not stimulated fermen-

tation research programs. Attempts have beenmade to improve the yields of phenazines andpyrrolnitrin.

Elander et al. (28) reported an example of arational approach to the selection of mutants ofP. aureofaciens which produce increasedamounts of pyrrolnitrin. Starting from the ob-servation that the addition of 1 g of D,L-trypto-phan per liter to the fermentation medium re-sulted in a doubling of pyrrolnitrin production,they isolated analog-resistant mutants synthe-sizing higher levels of tryptophan, thereby ob-viating the need to add exogenous tryptophan.A mutant resistant to 250 mg of 5-fluorotrypto-phan per liter produced twice as much pyrrol-nitrin as the parent strain without D-tryptophansupplementation. In a two-step mutant resistantto 1 g of 6-fluorotryptophan per liter, the levelof pyrrolnitrin in a nonsupplemented mediumwas three times that of the wild type andreached 150 mg/liter. Analog-resistant, high pro-ducing strains did not excrete tryptophan andincorporated less exogenous D,L-["4C]tryptophaninto pyrrolnitrin than their analog-sensitive par-ent. As uptake studies with both the D- and theL-isomers of tryptophan revealed no differencesbetween the parent and the analog-resistant mu-tants, the biochemical basis of analog-resistanceand a concomitant increase in pyrrolnitrin for-mation was thought to be due to a higher poolof endogenous tryptophan, the biosynthetic pre-cursor of pyrrolnitrin. In view of the versatileand efficient amino acid degradative pathwaysof pseudomonads (20), the production of aminoacid-derived secondary metabolites might be in-creased not only by interfering with the controlof the biosynthesis of an amino acid precursor,but also by imposing genetic blocks in the cata-bolic pathways responsible for its degradation.The effect of various nutritional factors on the

production of phenazines by pseudomonads and,more specifically, on the production of pyocy-anine by P. aeruginosa have been summarizedby Ingram and Blackwood (70) and MacDonald(88), respectively. Pyocyanine production by P.aeruginosa starts after the exponential growthphase. However, with the exception ofone report(44), the production of pyocyanine with suspen-sions of resting cells has not been achieved (69).The fact that media on which the organismgrows but does not produce pyocyanine havebeen found in many cases classifies pyocyanineand the phenazines in general as typical second-ary metabolites. On defined growth media con-taining glycerol as a carbon source, leucine oralanine or both as a nitrogen source, and con-trolled levels of Fe2", Mg2", and P04-, pyocy-anine yields of approximately 200 mg/liter have

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been achieved (88). The production of pyocy-anine required an optimal supply of Fe2" (>0.5mg/liter) and decreased when iron was thegrowth-limiting factor of the medium (79).

Several reports stress the importance of phos-phate limitation for directing the metabolism ofpseudomonads towards the production of phen-azines. Ingram and Blackwood (70) proposedthat the low phosphate levels reached in pro-duction media towards the end of the growthphase are instrumental in triggering pyocyaninesynthesis. Addition of high concentrations ofphosphate to the culture at this stage stoppedpyocyanine production, whereas addition of alimiting amount of phosphate led to an increasein cell mass and pyocyanine fornation. Similarobservations were made by Ingledew and Camp-bell (69), who suspended pregrown cells of P.aeruginosa in a phosphate-free 2-ketogluconatemedium. Pyocyanine synthesis was initiatedwhen the residual phosphate level had droppedbelow the limit of detection. It stopped when thecarbon source was used up and free phosphate,probably originating from ribosome degradation,appeared in the medium. Pyocyanine synthesisthus lends itself as a model for studying "phos-phate repression," a phenomenon affecting theproduction of various antibiotics (90).Another instance where phosphate repression

was studied is the production of hydrogen cya-nide, a degradation product of glycine exclu-sively formed by some species of Chromobacter-ium and Pseudomonas among procaryotes.Wissing (148) has proposed that bacterial cya-nogenesis from glycine proceeds by two oxida-tion steps involving flavoproteins, which convertthe C-N single bond of glycine to the C-Ntriple bond via an imino acid intermediate. Cas-tric (15) has demonstrated that hydrogen cya-nide biosynthesis in P. aeruginosa has the fol-lowing characteristics of secondary metabolism.The product was not formed during the lag andexponential phases of growth, active cyanoge-nesis occurred during the transition from expo-nential growth to the stationary phase, and lim-ited cyanogenesis was observed in the stationaryphase. Cyanide formation was of limited taxo-nomic distribution. Among 110 P. aeruginosaisolates, 74 produced hydrogen cyanide. Amongother Pseudomonas species tested only one offive strains of P. fluorescens was cyanogenic.Cyanide production by growing P. aeruginosahad a narrower temperature range than growth,required Fe3" concentrations above 0.6 mg/liter,and was inhibited by inorganic phosphate.

Inhibition of cyanogenesis by phosphate wasstudied in some detail by Meganathan and Cas-tric (93). Maximum hydrogen cyanide formationoccurred between 1 and 10 mM phosphate in a

growth medium containing glycine. Above andbelow this concentration range the rate and thequantity of cyanide production decreasedsharply, whereas growth was affected onlyslightly. Addition of chloramphenicol to a lowphosphate medium, prior to a phosphate shift-up triggering cyanogenesis, was inhibitory tocyanide production, indicating that protein syn-thesis is a prerequisite for phosphate-inducedcyanide formation. The demonstration of cya-nide production from glycine in a crude partic-ulate preparation from a Pseudomonas sp. (149)should allow the biochemical characterization ofthis process and ultimately the clarification ofthe role of phosphate in regulating the activityor synthesis, or both, of the enzyme(s) involved.Knowles (77), in a review on microbial cyanide

metabolism, has extended the similarity be-tween cyanide production and the production ofsecondary metabolites to the possible functionsof these compounds for the producing orga-nisms. He pointed out that cyanide excretioncould destroy or inhibit growth of neighboringorganisms, thereby conferring selective advan-tages on cyanogenic strains. In this contextWeinberg and collaborators (36, 144) have sug-gested that P. aeruginosa, like other microbialcells unable to differentiate, depends on success-ful completion ofsecondary metabolism for long-term survival in its natural environment. Thisview was based on the observation that phos-phate concentrations that inhibited pyocyaninefornation caused glycerol-grown cells to dieearly in the stationary phase (36). However, theexperiments supporting this interesting hypoth-esis are inconclusive because the survival ratesand the phosphate concentrations in the mediawere not compared with the quantity and thepattern of secondary metabolites produced. Fur-thermore, recent experiments by Turner (per-sonal communication) comparing the viability ofthe wild type and non-pigment-forming mutantsof P. phenazinium on phosphate-limited mediashowed that the wild type lost viability morerapidly than the pigmentless mutants aftergrowth was complete.

SUMMARY AND CONCLUSIONSThe first investigations on therapeutically

useful metabolites from fluorescent pseudomo-nads date back some 80 years. Among the con-siderable number of antibiotic substances thathave been isolated since then, only two (pyocy-anine and pyrrolnitrin), or possibly three (pseu-domonic acid), have reached the stage of prac-tical application. Reports on strain developmentprograms and on efforts to increase secondarymetabolite production are scarce, and most in-

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vestigations of secondary metabolites of pseu-domonads have been abandoned after the iso-lation of the pure product. With the exceptionof phenazine and pyrrolnitrin biosyntheses thereis little information available on the biosyn-thesis, genetics, and regulation of secondary me-tabolites from this group of microorganisms.

Nevertheless, it is important to integrate re-search on secondary metabolism into the rapidlyadvancing research on the biochemistry and ge-netics of Pseudomonas. The unicellular mode ofgrowth of pseudomonads, their nutritional ver-satility, the increasing knowledge on their bio-chemistry, and the availability of genetic tech-niques represent experimental advantages fre-quently missing in other producers of secondarymetabolites. Information on the production ofsecondary metabolites gained in studies withpseudomonads may be applicable to the morecomplex but industrially more important strep-tomycetes and bacilli. At the metabolic level thebiochemistry and regulation of amino acid bio-synthesis and of the multiple amino acid degra-dative pathways of pseudomonads are beingstudied intensively. However, the enzymes cat-alyzing the formation of amino acid-derived sec-ondary metabolites are not known nor are theregulatory mechanisms of the primary pathwayspermitting the feeding of the secondary path-ways. Integration of investigations on secondarymetabolite formation into research on aminoacid metabolism may lead to a better under-standing of the role of catabolic reactions in theformation of certain secondary metabolites andmay reveal general regulatory mechanisms af-fecting both primary and secondary metabolism(24).At the genetic level the various functions of

plasmids in pseudomonads attract much interest(18, 78, 147). Serious attention will undoubtedlybe given to the possibility that certain secondarymetabolites produced by pseudomonads areplasmid determined. Preliminary evidence indi-cates that some of the toxic amino acids pro-duced by pseudomonads are plasmid associated(62). If this is confirmed, studies in these orga-nisms may contribute to the analysis of plasmidinvolvement in the biosynthesis of secondarymetabolites. In view of the ideal growth prop-erties and the nutritional versatility ofpseudom-onads such investigations might also lead to theevaluation of these organisims as hosts for for-eign plasmids specifying antibiotic formation.

Finally, the low-molecular-weight phytotoxinsproduced by the phytopathogenic pseudomo-nads of the species P. syringae are of practicalinterest because they are considered responsiblefor the disease symptoms caused by the invadingbacteria in host plants (132). Studies on the

formation of these secondary metabolites thuswill contribute to our knowledge, and ultimatelyto the control, of certain plant diseases.

ACKNOWLEDGMENTSWe thank Rh6ne-Poulenc Industries for support of

R. M. during his stay at the Swiss Federal Institute ofTechnology, Zurich.We are indebted to D. Haas and J. M. Watson for

helpful discussions and to J. M. Turner for communi-cating unpublished data. The expert secretarial helpof Helene Paul is gratefully acknowledged.

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