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Annu. Rev. Microbiol. 1975.29:505-524. Downloaded from www.annualreviews.org Access provided by University of Manchester - John Rylands Library on 01/24/15. For personal use only.
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THE GENETICS OF DISSIMILARITY
+1668
PATHWAYS IN PSEUDOMONAS M L. Wheelis Department of Bacteriology, University of California, Davis, California
95616
CONTENTS INTRODUCTION.. ............................................. ................. Genetic Structure 0/ Pseudomonas . ...... ........................ ..........
507 PLA SMIDS .................................... . ....... .............. . . . ... . . . . . . 507 De/ect ive Phage .. ...................... . ... . ....... . . . ........ .......... Degradotive Plasmids. .... . .... . . ........... . ......... . . . . . .. . . . . . . .. . . . 510 The CAM plasmid.. .... ....................... ....................... 510 The OCT plasmid.. .................. ................. ........ .. ..... 511 513 The SAL plasmid .................................................... 514 The NAB plasmid.................................................... The TOL plasmid.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514 Plasmids as Sex Factors.. ................................................ 515 516 Summary. ............................................................. CHROMOSOMAL CLUST ERING. ................................................. RE GULATION OF GENE EXPRE SSION........................................... The The The
ami System ........................................................ hut System.. ...................... ................................. catBC Syst e m ..... .... . ........... .................................
507
516 517
517 518 518
EVOLUTIONARY CONSIDERATIONS OF GENE TIC EXCH ANGE................... CONCLUDING REMARKS..
505
.....................................................
519 522
INTRODUCTION
The vast majority of microbial genetic investigations have been conducted using markers that interfere with the biosynthetic metabolism of the test organism. Very few have been concerned with the genetics of dissimilatory pathways, despite the fact that the two verified models of gene control at the molecular level [i.e. negative control of the lac operon (41) and positive control of the ara operon (67), both in Escherichia coli] derive from investigations of this kind of pathway. One of the major reasons for this neglect is target size; the number of genes, mutation in which will result in an auxotrophic phenotype is obviously much greater than the number of genes that will affect one particular catabolic pathway. Thus if one aims to determine the gross genetic structure of an organism, auxotrophic markers are 505
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usually initially employed. Such an approach is based on the assumption that genes encoding biosynthetic function are distributed randomly over the chromosome (and over any other linkage groups, all of which, together with the chromosome, com prise the genophore of the organism). One of the theses of this essay is that such an assumption is not warranted. In addition to the lactose and arabinose systems, a few other catabolic pathways have been studied in the enteric bacteria. These include histidine (69), proline (27), acetoacetate and butyrate 1(55), and a variety of sugars. As is obvious from this list, all these substrates are closely related to or identical with end products or intermedi ates of biosynthetic pathways. The reason for this becomes clear if the metabolic versatility of the enteric bacteria is examined: Gutnick et al (33) found that Sal monella typhimurium LT-2 was capable of utilizing 73 compounds out of about 600 tested as sole source of carbon and energy, and most of these compounds share the above-noted relationship to compounds of biosynthetic metabolism. In marked contrast to the enteric bacteria, the pseudomonads display great versatility in terms of the organic compounds that support growth (70); a screening of only 145 compounds allowed the identification of over 100 that could be utilized by a single strain of Pseudomonas multivorans. Although slightly less versatile, the strains utilized for genetic studies (principally Pseudomonas putida and P. aeruginosa) are nevertheless capable of degrading a wide variety of organic compounds, many of which are quite unusual (e.g. camphor, naphthalene, and aromatic acids). Rates of growth supported by some of these compounds are often faster than those supported by compounds such as glucose. These pseudomonads are common soil saprophytes (including the facultative pathogen, P. aeruginosa), and one report (63) indicates that their abundance in soil is directly correlated with proximity to a source of organic carbon. Apparently, the ubiquity of soil pseudomo nads reflects their ability to utilize almost anything as a source of carbon and energy, rather than any particular ability to maintain viability when not actively growing or to compete successfully for readily metabolized compounds. Such a pattern of selective forces is in striking contrast to those that can be deduced to have guided the evolution of the enteric bacteria, in which selection seems to have favored organisms with exceptionally precise control of cellular metabolism. This precision, which presumably optimizes growth rate by minimizing energy waste, has been accomplished in part by bringing all genes that function together into close proximity on the chromosome and subjecting them to control by a single set of regulatory genes. Thus, a feature of the genetic map in enteric bacteria is the clustering of functionally related genes (64, 72). Additional economy is gained by feedback inhibition, which operates to prevent the functioning of a pathway when its end products are available. In the pseudomonads, precise control of enzyme synthesis seems to have been less important. Although feedback inhibition appears to function with efficiency compa rable to that in enterics (indicating the importance of regulating pathway activity), repression of biosynthetic enzymes seems much less precise than in the enterics (24). For instance, in pseudomonads the pathways of tryptophan (25), arginine (40), isoleucine and valine (47), and pyrimidines (39) all contain at least one enzyme whose synthesis is constitutive. To date, no biosynthetic pathway in this genus has
PSEUDOMONAS
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Annu. Rev. Microbiol. 1975.29:505-524. Downloaded from www.annualreviews.org Access provided by University of Manchester - John Rylands Library on 01/24/15. For personal use only.
been found to be fully repressible. This is reflected in the dramatically more scattered arrangement of the genes of any given biosynthetic pathway on the Pseudomonas chromosome (36). Very probably, selection in a soil environment often favors organ isms able to grow at a moderate rate on many compounds over those that grow very quickly on only a few. Genetic Structure of Pseudomonas
The Pseudomonas genophore is composed of a chromosome and perhaps any num ber of a varied collection of plasmids. The chromosome has been sized variously as 4 X 109 daltons in P. jiuorescens, P. stutzeri, and P. oleovorans and 7 X 109 daltons in P. aeruginosa by renaturation kinetic measurements (I), and as 2.4 X 109 daItons (in P. aeruginosa) by a combination of physical methods (J. M. Pemberton, personal communication). This discrepancy is hard to reconcile because there is no obvious reason to fault either investigation. One rather unlikely, but nevertheless tantalizing, possibility is that Pseudomonas has multiple chromosomes. More likelY, either there is a flaw in one of the determinations or Pseudomonas is very heterogeneous in terms of its DNA content (no strain was common to both investigations). Based on analogy to other prokaryotes, the 2.4 X 109 dalton value appears to be more proba ble. At least some strains of Pseudomonas harbor one or more plasmids in addition to the chromosome. These include fertility factors and R factors (see 36 for a review), a mercury resistance factor (15), plasmids coding catabolic enzymes (dis cussed below), and cryptic plasmids (32, 46, 56). One report indicates that P. aeruginosa strains gradually lose plasmids after isolation and upon laboratory cul ture (32). If, as suggested below, genetic transfer occurs in natural environments at a frequency sufficient to have had a -decided genetic effect upon evolution in the genus, it seems reasonable to consider the genetic structure of the population. As with eukaryotic populations, the individual is the direct target of selective forces, but the long-term effect mediating evolutionary change is variation in the genetic structure of the population; crudely, this means variation of allele frequencies. The Pseudomo nas genophore can be regarded as consisting of three classes of genes: (a) essential, (b) often useful, and (c) occasionally useful (59). Genes of the first two sets are most likely all located on the chromosome. Genes in the last set, including those for catabolic pathways, may be located on the chromosome of one strain but be plasmid borne or absent in others. Continual interstrain transfer of these plasmids assures a constant reassortment of nutritional types; transfer of chromosomal loci insures that combinations not achievable by plasmid transfer will also arise. PLASMIDS Defective Phage
The first reliable transductional system in P. pUlida was described by Gunsalus and his co-workers (16), utilizing two wild-type strains. One of these, now called PRS I, was isolated as A.3.12 by Stanier from a lactate enrichment and is the strain used
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in his laboratory for extensive studies of the biochemistry and regulation of aromatic acid catabolism. It has been designated the type strain of P. putida (70). The other was isolated in Gunsalus's laboratory from a camphor enrichment as strain C l. This strain is now designated PpG1 (or PpG2, a colony morphology mutant of PpG 1), but was for a short while termed PUGI (or 2). The phage employed was pf16 or its host-range mutant, pf16.h2. The latter is capable of plating on either PRSI or PpG2, and like the wild type is capable of mediating generalized transduction. Initial studies (l6) indicated that interstrain transduction between PRSI and PpG2 can be accomplished. The selection was for growth of PpG2 with mandelate, a compound normally unable to support the growth of this strain (Figure I). Both strains are capable of growth with benzoate; but PpG2 is unable to utilize D- or L-mandelate or benzoylformate. Chakrabarty et al (16) speculated that this is because of a complete lack of the genes involved. This contention is supported by D{-)mandE!late
! ! !
L{+)mandelate
benzoylfo"mate
be
nz
aldehyde
!
benzoalle
p-hydroxybenzoate
+ .. l+
catechol
co
protoC
!
'Y-carboxymuconolactone
(+) muconolactone
MI(catCl
J
Chuate
/l-carboxy-c,s,c,s-muconate
C/s,c,s muconate MLE (catB)
+
-----...
�
/l-ketoadipate enol lactone
!3
- , ,
ketoadipate
/l-ketoadipyl-CoA
+
acetYl-Co A + s u cci nate
Figure 1
The {3-ketoadipate pathway for aromatic acid dissimilation. Gene and enzyme
designations are as used in the text. Bold arrows indicate enzymes common to PRS I and PpG2, light arrows those of PRSI only.
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the following findings: (a) no activity for any of the four assayed enzymes can be found in PpG2; (b) there is no material in PpG2 that gives an immunological cross-reaction with antibodies toward two PRSI mandelate enzymes; and (c) when the donor strain is deficient in mandelate dehydrogenase, so are all PpG2 mdl+ transducants (i.e. PpG2 cannot complement an mdlB mutation). Further investigation of this system (17) established that it is unusual in that the mechanism of establishment of mdl+ PpG2 strains appears to be via lysogenization by a defective phage particle carrying the mandelate genes. The evidence underlying this assertion can be summarized as follows: (a) the buoyant density of most mdl carrying transducing particles is intermediate between that of the pfu peak and the peak of met-carrying particles, a result consistent with their carrying a mixture of phage and bacterial DNA (which differ substantially in buoyant density). The met carrying particles are assumed to contain only bacterial DNA; (b) upon UV irradia tion and superinfection, lysates are formed that transduce mdl genes at elevated frequency; (c) many of the transductants are immune to vegetative superinfection by pf l6.h2; (d) the mdl genes segregate at a high spontaneous frequency (about 10-3 per bacterial division), which can be increased by UV irradiation; and (e) mdl+ PpG2 transductants show a satellite band of DNA on CsCI gradients inter mediate between the band of the main bacterial DNA and that of pfl6.h2 DNA; the satellite band is not shown by wild-type PpG2 or cured transductants. The evidence thus seems compelling that the mandelate+ phenotype in these transduc tants is a consequence of their acquisition of a (presumably extrachromosomal) element composed of (presumably covalently linked) phage and bacteria DNA. Calculations indicate that the transducing particles probably contain 35-70% bac terial DNA. There is conflicting evidence concerning the number of copies of this element (termed pfdm) per cell. Initial assays of the level of enzyme activity (16) suggest that there is one copy per chromosome, since they are about equal in the transduc tants and in the donor. Later studies (17), however, give 3-6-fold higher levels of activity in the transductants than in PRS1, suggesting the possibility of multiple copies. I have projected Figure 5 of Chakrabarty & Gunsalus (17), determined the relative areas of chromosomal and satellite peaks, and concluded that pfdm com prises 3.9% of the DNA of these transductants. If we assume a mol wt for the transducing fragment equal to that of the wild-type phage (9 X 107 daltons) (J. F. Niblack, personal communication) and a mol wt of 2.4 X 109 for the chromosome, one copy of pfdm per chromosome should represent 3.8% of the DNA. Thus, it is likely that pfdm and the chromosome are present in equal numbers. Should the chromosome of P. putida be found to be much larger than the above value, we should conclude that a larger number of copies of pfdm are present. It should be noted that phage pfl6 is a virulent phage, apparently totally incapable of establishing lysogeny and capable only of generalized transduction. However, these findings indicate that it is able to recombine with the chromosome to produce transducing particles, which appear to be of the type mediating specialized transduc tion (51). Although generalized transducing particles in Salmonella phage P22 have been reported to contain some phage DNA [(65); but cf (29) for a conflicting view],
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WHEELIS
there is no evidence that such particles can establish lysogeny. Phage Pi, however, seems to be quite similar to pfl6 in that PI dl particles seem to have subst,antially the same structure as pfdm and are selected in crosses in which the recipient lacks the region being transduced (45). Thus, lack of homology may select for transduc tants by repliconation (20) rather than recombination. Because there are a number of nutritional differences between the two strains involved here (70), such specula tion is open to test. Degradative Plasmids The other major class of plasmids considered in this review is that comprised of plasmids carrying genes that specify enzymes of dissimilatory pathways. Chakrab arty has suggested (II) that these be termed degradative plasmids, and that nomen clature is adopted here. Five pathways are claimed to be specified by genes with plasmid location in one or more strains of Pseudomonas: camphor, octane, naphtha lene, m-tolute, and salicylate. They are discussed individually. Rheinwald et al (58) have presented evidence that the path way via which D or L-camphor is converted to isobutyrate is coded by plasmid-borne genes. The evidence rests on segregation frequencies and upon the self-transmissible nature of the cam genes. There are, however, conflicting reports concerning trans ductional linkage of these genes. Rheinwald et al have presented evidence indicating all the genes involved in the conversion of camphor to isobutyrate that were tested are cotransducible at high frequency. Thus, camA, camB, and camC (which code the three subunits of the first enzyme of the pathway) are closely linked to camD (second enzyme) and to camG (one of three subunits of the third enzyme). Several other markers, not further (:haracterized biochemically, that render the cell unable to utilize camphor but still able to ,use isobutyrate were shown also to cotransduce with camABCDG. The authors assert that none of these genes cotransduces with genes that prevent the utilization of isobutyrate. For this and other reasons (outlined below), Rheinwald et al propose that the cam genes are located on a linkage group separate from that (presumably the chromosome) occupied by the genes involved in isobutyrate catabolism (ibu) . However, an earlier report (16) asserted that not only did cam-l (hydroxylase negative, therefore probably a comA, -B, or -C muta tion) cotransduce at high frequency with cam-2 (biochemically uncharacterized), but that both of these gene!; cotransduced with /pa-I. The latter marker is lihked to some genes governing tryptophan biosynthesis and is almost certainly chromoso mal. A possible explanation for this discrepancy is suggested below. Despite the conflicting linkage data, the evidence supporting an autonomous state for the cam genes appears good. First, the spontaneous rate of mutation to cam is high (about 10"-4 per cell division) and can be enhanced by mitomycin C at sublethal concentrations (to close to one per cell division). These cam mutants do not revert at detectable frequencies; they lack all assayed enzymes of the camphor pathway and fail to yield cam+ transductants when used as either donors or recipi ents. This suggests that these genes are resident on a plasmid too large to be transduced by pfl6 in its entirety (i.e. probably >9 X 107 daltons). Second, the THE CAM PLASMID
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ability to utilize camphor can be transferred either to a spontaneous cam- mutant of PpG1 or to other strains of Pseudomonas-including several P. aeruginosa strains and one P. fluorescens strain-by mixing donor and recipient cultures. Third, the CAM plasmid is incompatible with other known plasmids, including one containing part (at least) of pfdm plus additional chromosomal material (58) and the plasmid (OCT) carrying genes for octane dissimilation (14). Chou et al (19) have isolated heat-sensitive mutants that apparently fail to repli cate CAM at the restrictive temperature. The mutation is chromosomal and does not affect the replication of OCT. Loss of CAM does not commence until about eight generations after temperature shift, which implies either that there are multiple copies of CAM present (unlikely, as several hundreds would be required) or that the block affects the synthesis of a cellular component present in excess. Pseudomonas pu tida dissimilates octane via octanol, oc tanaldehyde, and octanoic acid (2). The octanoate is presumably further metabol ized by f3-oxidation. At least two inductive events are involved in allowing growth on octane, as octanol does not induce the octane monooxygenase, yet does serve as a carbon and energy source. The monooxygenase is composed of three proteins (57), and as the octanol and octanaldehyde dehydrogenases are distinct (2), at least five structural genes are involved in the conversion of octanol to octanoate. The evidence summarized below indicates that at least some of these genes reside on an extra chromosomal element. The introduction of the CAM plasmid from Cl strains into the strain used by Baptist et al (2) [called P. oleovorans (2, 14, 57), but actually probably P. putida (70)] can be accomplished at low frequency by conjugation (see above), but the exconju gants become unable to grow with octane (14). Most (>90%) are also unable to grow with octano!' If octanol-negative exconjugants are subsequently cured of the CAM plasmid by mitomycin C treatment (see below) or are selected for spontaneous octanol positive strains, the CAM plasmid is lost and the cells simultaneously become capable of growth on octano!' They remain octane negative and do not revert. It thus appears that at least part of the octane hydroxylase is coded by a plasmid designated OCT that is incompatible with CAM, and that the chromosome carries the octanol and octanaldehyde dehydrogenases. The failure of most CAM+ OCT- exconjugants to grow with octanol was attributed (14) to repression by the CAM plasmid of the expression of the chromosomal dehydrogenase genes. The fact that some of these exconjugants are octanol positive has not been satisfactorily explained. In addition to being incompatible with CAM, OCT is defined as a plasmid on the basis of its curability and transmissibility. The rate of curing of OCT in the presence of sublethal mitomycin concentrations goes up to about 7% from ,
caICBR
,
mdlC
!
omiER
�
ben
/
P. pulido
Figure 2
Comparison of gene arrangement in P. pulida and P. aeruginosa. Breaks indicate
lack of demonstrated linkage. For clarity, the fragments of the P. aeruginosa map are arranged to coincide with
the P.
pUlida map; such coincidence has
not
been experimentally demon
strated. Gene designations: ben, mdl, cat, pob, ant, and pea for the J3-ketoadipate pathway
42, 61, 62, 74-); pal for phenylalanine (74); pac for phenylacetate (74); (43, 74); ami for amidase (6); and net for nicotinate (44).
(see Figure 1; and for histidine
hut
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quite differently in the two species (60). Gene arrangement in the pseudomonads, as in the enterics (64, 72), seems to be a fairly stable property. This region represents a minimum of 14 operons in P. putida (44). The end-to-end span is 9% of the bacterial chromosome [based on distances derived from pf16. h2-mediated transductions, analyzed by the method of Wu (78), and assuming a transducing fragment length of 9 X 107 daltons, with a chromosome of 2.4 X 109 daltons]. This P. putida map includes every mutation blocking a catabolic pathway that we have mapped; thus, to the limits of present resolution, every chromosomal gene with catabolic function is located in this region. Such clustering cannot be random (52). At present, it is unknown whether there are genes with noncatabolic function interspersed among the genes shown. There is room for at least 300 addi tional genes between ben and pobA, and it would be extremely interesting to know what functions they specify. Our failure to isolate large deletions in this area (M. L. Wheelis and B. J. Leidigh, unpublished observations) suggests that there are essential functions coded between the identified catabolic genes. I thus surmise, without any firm experimental support, that in Pseudomonas genes with biosyn thetic and other "essential" functions are randomly distributed over the chromo some (i.e. they are not excluded from the region under discussion) but are virtually absent from extrachromosomal linkage groups, whereas genes with catabolic func tion have a decidedly nonrandom organization on the chromosome and are fre quently found on other linkage groups. REGULATION OF GENE EXPRESSION
Regulation at the genetic level has been studied in detail in only three systems in Pseudomonas: the hur, carBe, and ami loci. Of these, one appears to be negatively controlled and one appears to be under positive control; the regulation of the third system is unclear at this time. The ami System
The regulation and genetics of the aliphatic amidase of P. aeruginosa have been studied by Clarke and her co-workers. This inducible enzyme converts acetamide, glycollamide, or propionamide to corresponding carboxylic acids. Selection for use of formam ide (a weak inducer and poor substrate) as sole source of nitrogen has allowed the ready isolation of constitutive mutants. In addition, the selection from parent strains constitutive for amidase production of strains able to use higher amides (butyra,mide, bon source led 'to an elegant series of studies of experimental evolution (5, 8, 9). Transductional mapping of the genes involved has indicated very tight linkage of amiR (the regulator gene) and amiE (the amidase structural gene) (6), and amiR has been used as an outside marker for the ordering by three factor crosses of a number of amiE lesions, including some that involve changes in the substrate specificity of the enzyme (3). The ami loci appear to be transductionally unlinked to the (presumably structural) genes, mutation in which abolishes synthesis of isocitrate lyase, malate synthase, citrate synthase, or acetic thiokinase (68).
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WHEELIS
All of the published data is compatible with amiE being subject to classical negative control by a repressor protein, the structural gene for which is amiR. A recent abstract (30), however, suggests the possibility of positive control, based on (a) the lack of any temperature-sensitive constitutives (i.e. inducible at low tempera ture, constitutive at high temperature) and (b) the isolation of two strains that are constitutive at low temperature and unable to synthesize amidase at high tempera ture, and whose amidase appears identical to wild type by several criteria. Although neither of these considerations is in direct conflict with the classical model for negative control, they are somewhat more consistent with the system's being subject to positive control. This is especially true of b, which would, in a negative control model, demand that a single mutation abolish the function of the inducer-binding site on the repressor protein (at least at high temperature) and render operator binding cold sensitive. While there is nothing intrinsically improbable about such a possibility, it is a mutant type not previously described, even in the lac system of Escherichia. It thus seems prudent to await the results of further experimentation before drawing conclusions as to the nature of the control of the P. aeruginosa aliphatic amidase. The hut System
Histidine is dissimilated by P. putida via a five-step pathway (71), the first four enzymes of which are specified by the genes hutHUIF. A study of the control of this system (43) indicated that although the detailed biochemistry differs from that in the enterics, the regulation shows some striking similarities. First, the inducer of all four enzymes is the same (urocanic acid, the first intermediate). Second, the system appears to be under negative control exerted by the product of hutC, which is located in the midst of thl� structural genes of the pathway. Although the evidence is solid for the enteric bacteria (e.g. see 69), in P. putida the only real evidence for negative control is the infrequency of pleiotropic negative mutants (one out of about 40) and the ease with which constitutives were isolated in hutC. [The latter were selected by alternating between culture with histidine and glutamate, the product of the pathway, thereby selecting organisms that commenced growth without a lag. Such selection, first used by Cohen-Bazire & Jolit (23), should be of general utility when few inductive events occur between substrate and product.] Thus, although there is really no good evidence for classical negative control in the hut system, neither is there evidence basically inconsistent with the (probably at least two) hut operons being subject to such control; however, the superinducibility of both hutC mutants is curious. The catBe System
Although all parts of the I�-ketoadipate pathway have been studied in P. putida in great physiological detail (see 50 for a review), only one of the several presumed "operons" has been examined with any genetical detail (73, 77). This is the catBC system, which codes two f'nzymes involved with catechol dissimilation (Figure 1): muconate-lactonizing enzyme (MLE) and muconolactone isomerase (MI). The in ducer, cis,cis-muconate, is the substrate of the first of the two enzymes (MLE) and
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also the inducer of catechol oxygenase (CO) (the enzyme that produces it), although CO induction is noncoordinate with that of MLE and MI (49). About a quarter of the negative mutants isolated for this system were pleiotropically negative, i.e. MLE-MI-. Deletion mapping (73) established that all of these pleiotropic-negative mutations clustered at one end of the catB gene. The two likely explanations are that either these represent mutations in a gene with positive regulatory control function or they are polar nonsense mutations in catB. Evidence against the latter is that mutagen-forced revertants of a small deletion with pleiotropic effects contain an MLE with identical immunological properties and rates of thermal denatura tion (77). The original deletion therefore probably did not enter catB. In addition, some of these revertants were weakly or strongly constitutive for MLE(catB), MI(catC), and CO(catA). A single mutation thus is sufficient to change the nonin ducible phenotype to constitutivity, which strongly suggests a regulatory gene (termed catR). This gene presumably produces a product that turns on the catBC "operon" and catA (presumably at separate sites). Two curious facts remain unex plained. First, why is CO constitutive in the constitutive revertants of catR-, yet normally inducible in all catR- strains? And second, why are no catR- mutants or revertants temperature sensitive [if the catR product were a protein, one would expect that many of the primary and secondary mutants would be temperature sensitive (e.g. 38)]? EVOLUTIONARY CONSIDeRATIONS OF GENETIC EXCHANGE
In addition to the discussion here, the interested reader is referred to the excellent discussions of the evolution of catabolic pathways in (4, 21, 35). It was fashionable for a time to consider that genetic exchange among bacteria was a laboratory artifact (e.g. 34) and of no consequence in natural populations. This view is now changing, and I discuss it with particular regard to Pseudomonas. The fact that pseudomonads are now recognized as carrying a variety of transmissible plasmids argues compellingly for the operation of transfer functions in nature, for we would expect such functions to have been lost if not selected. The possibility exists, of course, that what we term transfer functions have in reality other roles and that transfer is simply a side effect of these other functions; until there is some evidence for such pleiotropy, however, the simplest approach is to assume that the presence of self-transmissibility reflects the selection of such a phenotype. We con clude that genetic exchange, at least of plasmids, is important in natural populations. It should be noted that the spread of R factors among the enteric bacteria provides a striking and widely known example of the natural importance of plasmid transfer in another group of gram-negative bacteria, although one with a drastically different ecology. A similar conclusion about the importance of genetic transfer of chromosomal loci derives from consideration of the possible roles of chromosomal clustering. The degree to which genes with catabolic function are clustered, clearly in a supraoper onic fashion, defies explanation on the basis of shared elements of control of gene
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expression. We therefore proposed (74) that the selective advantage was to allow the simultaneous transfer of the genes involved. This argument for the separate operons of a single biochemical pathway is clear, because any one part of a pathway may be physiologically functionless without the rest. For separate pathways, the argu ment is less clear but can still be applied: transfer of a genetic region containing the determinants of more than one catabolic pathway would allow the recipient of this region to gain several new pathways in a single exchange event [see (44) for addi tional treatment of both this point and the role of duplication in evolution (discussed below)]. Alternatively, the clustering of genes involved in different catabolic path ways could have arisen from selection involving gene dosage effects. Thus, if the probability of gene transfer in wild populations is related to gene dosage, those genes whose transfer is most advantageous might come to be located in proximity to the origin of replication. Other effects of position relative to the initiation site for replication, such as raising (or lowering) the maximal level of gene expression, are less likely to have been selected because they are obtainable more simply and precisely by other means, such as variation in promoter specificity. This argument for the existence of genetic exchange among natural populations of bacteria says nothing about its actual frequency. It simply asserts that such exchange has been an important force in the evolution of the pseudomonads. In fact, because genetic exchange must be a second-order reaction at best and because natural Pseudomonas population densities are usually probably rather small (at least compared to densities of enterobacterial populations), the actual frequency of ex change must be rather low. A naive expectation would be that modification and restriction systems might be a liability because they could reduce this frequency to the level of nonexistence. In fact, at least as far as episome transfer is concerned (10), restriction does not seem to occur in strains of fluorescent pseudomonads, with the exception of P. aeruginosa. P. aeruginosa is a quite precisely definable and homoge neous species in terms of both phenotypic traits and DNA-DNA homology group ings, in contrast to the other species of pseudomonads, which are relatively heterogenous (53). This suggests that P. aeruginosa has been genetically insulated because of its restriction system, at least relative to other pseudomonads, among which there has been at least some interspecific exchange. Indeed amston (54) has argued from protein primary sequence data for relatively recent intergeneric genetic exchange (between Acinetobacter and Pseudomonas putida). Assuming, then, that genetic exchange does occur between bacteria in the wild at a rate sufficient to allow selection of various gene arrangements, it is possible to construct a model for the evolution of catabolic pathways that avoids a paradox in the Horowitz (37) "retroevolution" theory as applied to catabolic pathways. Horo witz postulated that the sequential steps in a biosynthetic pathway possess a filial relationship to each other, in that the genes coding the enzymes involved are derived one from another by duplication. Although this idea is fairly generally accepted for biosynthetic pathways, its application to the evolution of catabolic pathways has encountered some difficulties (e.g. see 35). Most of the objections (impermeability of intermediates, heterogeneity of the chemistry of sequential reaction steps, etc) seem to me to apply with equal force to both catabolic and anabolic pathways. There .
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521
is, however, one major difference between the two kinds of pathways, and that difference lies in the effect that the relative rates of duplication and recombinational loss of duplicated material will have (44). The rate of recombinational loss, in every system studied to date, is much higher than the rate of duplication; thus a duplicated region will be lost before there has been sufficient divergence between the two copies to both change the function of one and render the two dissimilar enough to lower the frequency of recombinational loss. The difference between biosynthetic and dissimilatory pathways with regard to this argument lies in the differences in inten sity and duration of selection that will be applied. Selection for a biosynthetic capability could quite easily have been continuous and strong, and in the systems studied to date, continuous selection can maintain a duplication indefinitely. The situation is different with respect to catabolic pathways, at least later in evolutionary history when the vast array of catabolic pathways characteristic of modern soil saprophytes presumably evolved. In this case, selection was almost certainly inter mittant, and the period between selective intervals was probably relatively long, especially for some of the more exotic pathways. Although the initial stages in the evolution of catabolic metabolism may have involved duplications (indeed the selective forces would probably have approached those guiding the emergence of anabolic capability), we have proposed (44) that later diversification of catabolic functions involved divergent evolution of strains, fol lowed by genetic exchange between them to yield recombinants that possessed the abilities of both parents. The end result is the same as in the case of duplication and divergence; the only difference being that genetic exchange allows the two diverging genomes to be protected against recombinational loss of duplicated regions because they are in different organisms. It should be noted that although I have accepted the likelihood of retroevolution both as the mechanism by which anabolic pathways evolved and as being possibly implicated in the early stages of development of catabolic pathways, there is no intellectual necessity for this. Gene exchange and the selective forces arising therefrom provide a formal alternative to retroevolution at all stages of biological evolution. One of the attractions of the retroevolution hypothesis is that it readily explains the genesis of operons, of which two steps can be envisaged: (a) the genes to be included must be brought into close proximity and (b) they must be subjected to common control. Presuming retroevolution involved principally tandem duplica tions, the first requirement is automatically fulfilled. If we abandon the retroevolu tion hypothesis for catabolic pathways, we must assume that the genes of an evolving pathway would be scattered on the chromosome. As we have seen, genetic transfer can provide a selective pressure for the clustering of genes with related function independently of shared control elements and may thus have been instru mental in forcing gene arrangements that later permitted the formation of operons (74). The same argument can, of course, be applied to the generation of plasm ids. Most dissimilatory plasmids probably could not have grown by gradual accretion of genes from the bottom up, because of the rarity, instability, or inability to cross the cell membrane of biochemical intermediates. Thus, acquisition of plasm ids probably
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confers no selective advant2lge unless they carry most or all of the determinants of a pathway. Therefore, on th,! assumption that most of such plasmid-borne pathways are ultimately derived from chromosomal genes, it seems likely that chromosomal clustering is a necessary intermediate in the genesis of dissimilatory plasmids. If such plasmids are indeed derived by recombinational excision from the chromo some, at least some of those more recently formed may in fact be able to reintegrate with the chromosome. Degradative plasmids may thus be in equilibrium between autonomous and integrated states. Such an equilibrium could explain the anomalous linkage results obtained for the CAM plasmid (see above) and is also consistent with the strain differences with regard to the genes for naphthalene dissimilation (plas mid-borne or chromosomal, depending upon strain) and with the ability of CAM to mobilize the chromosome. CONCLUDING REMARKS
Genes that specify enzymes with catabolic function in Pseudomonas appear in one of two distinctive linkage modes: they either are found in a small region of the chromosome or else are borne on plasmids. I interpret both kinds of topology as having been selected for their facilitation of genetic transfer. This sytem is of considerable interest because it may assist materially in the elucidation of the importance of both genetic recombination in the evolutionary history ofprokaryotes and the genetical structure of microbial populations in the wild. In my view, genetic exchange events play a crucial role in determining the genetic structure of such populations over both short and long time periods. In the short run, the spread of infectious plasmids can determine the extent to and rapidity with which a microbial population can degrade complex organic compounds; over longer periods of time, the evolution of novel abilities may depend upon recombination events. From a practical standpoint, an operative genetic approach to catabolism in a nutritionally versatile group of soil saprophytes may be immeasurably valuable in the construc tion of microbial strains capable of degrading some of the synthetic and noxious chemicals that man inflicts on the biosphere in increasing amounts. Even if such artificial strain construction proves not to be a feasible solution, the understanding of microbial population dynamics in nature is essential to sane environmental policy determination. Literature Cited 1.
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All rights reserved
THE GENETICS OF DISSIMILARITY
+1668
PATHWAYS IN PSEUDOMONAS M L. Wheelis Department of Bacteriology, University of California, Davis, California
95616
CONTENTS INTRODUCTION.. ............................................. ................. Genetic Structure 0/ Pseudomonas . ...... ........................ ..........
507 PLA SMIDS .................................... . ....... .............. . . . ... . . . . . . 507 De/ect ive Phage .. ...................... . ... . ....... . . . ........ .......... Degradotive Plasmids. .... . .... . . ........... . ......... . . . . . .. . . . . . . .. . . . 510 The CAM plasmid.. .... ....................... ....................... 510 The OCT plasmid.. .................. ................. ........ .. ..... 511 513 The SAL plasmid .................................................... 514 The NAB plasmid.................................................... The TOL plasmid.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514 Plasmids as Sex Factors.. ................................................ 515 516 Summary. ............................................................. CHROMOSOMAL CLUST ERING. ................................................. RE GULATION OF GENE EXPRE SSION........................................... The The The
ami System ........................................................ hut System.. ...................... ................................. catBC Syst e m ..... .... . ........... .................................
507
516 517
517 518 518
EVOLUTIONARY CONSIDERATIONS OF GENE TIC EXCH ANGE................... CONCLUDING REMARKS..
505
.....................................................
519 522
INTRODUCTION
The vast majority of microbial genetic investigations have been conducted using markers that interfere with the biosynthetic metabolism of the test organism. Very few have been concerned with the genetics of dissimilatory pathways, despite the fact that the two verified models of gene control at the molecular level [i.e. negative control of the lac operon (41) and positive control of the ara operon (67), both in Escherichia coli] derive from investigations of this kind of pathway. One of the major reasons for this neglect is target size; the number of genes, mutation in which will result in an auxotrophic phenotype is obviously much greater than the number of genes that will affect one particular catabolic pathway. Thus if one aims to determine the gross genetic structure of an organism, auxotrophic markers are 505
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usually initially employed. Such an approach is based on the assumption that genes encoding biosynthetic function are distributed randomly over the chromosome (and over any other linkage groups, all of which, together with the chromosome, com prise the genophore of the organism). One of the theses of this essay is that such an assumption is not warranted. In addition to the lactose and arabinose systems, a few other catabolic pathways have been studied in the enteric bacteria. These include histidine (69), proline (27), acetoacetate and butyrate 1(55), and a variety of sugars. As is obvious from this list, all these substrates are closely related to or identical with end products or intermedi ates of biosynthetic pathways. The reason for this becomes clear if the metabolic versatility of the enteric bacteria is examined: Gutnick et al (33) found that Sal monella typhimurium LT-2 was capable of utilizing 73 compounds out of about 600 tested as sole source of carbon and energy, and most of these compounds share the above-noted relationship to compounds of biosynthetic metabolism. In marked contrast to the enteric bacteria, the pseudomonads display great versatility in terms of the organic compounds that support growth (70); a screening of only 145 compounds allowed the identification of over 100 that could be utilized by a single strain of Pseudomonas multivorans. Although slightly less versatile, the strains utilized for genetic studies (principally Pseudomonas putida and P. aeruginosa) are nevertheless capable of degrading a wide variety of organic compounds, many of which are quite unusual (e.g. camphor, naphthalene, and aromatic acids). Rates of growth supported by some of these compounds are often faster than those supported by compounds such as glucose. These pseudomonads are common soil saprophytes (including the facultative pathogen, P. aeruginosa), and one report (63) indicates that their abundance in soil is directly correlated with proximity to a source of organic carbon. Apparently, the ubiquity of soil pseudomo nads reflects their ability to utilize almost anything as a source of carbon and energy, rather than any particular ability to maintain viability when not actively growing or to compete successfully for readily metabolized compounds. Such a pattern of selective forces is in striking contrast to those that can be deduced to have guided the evolution of the enteric bacteria, in which selection seems to have favored organisms with exceptionally precise control of cellular metabolism. This precision, which presumably optimizes growth rate by minimizing energy waste, has been accomplished in part by bringing all genes that function together into close proximity on the chromosome and subjecting them to control by a single set of regulatory genes. Thus, a feature of the genetic map in enteric bacteria is the clustering of functionally related genes (64, 72). Additional economy is gained by feedback inhibition, which operates to prevent the functioning of a pathway when its end products are available. In the pseudomonads, precise control of enzyme synthesis seems to have been less important. Although feedback inhibition appears to function with efficiency compa rable to that in enterics (indicating the importance of regulating pathway activity), repression of biosynthetic enzymes seems much less precise than in the enterics (24). For instance, in pseudomonads the pathways of tryptophan (25), arginine (40), isoleucine and valine (47), and pyrimidines (39) all contain at least one enzyme whose synthesis is constitutive. To date, no biosynthetic pathway in this genus has
PSEUDOMONAS
507
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been found to be fully repressible. This is reflected in the dramatically more scattered arrangement of the genes of any given biosynthetic pathway on the Pseudomonas chromosome (36). Very probably, selection in a soil environment often favors organ isms able to grow at a moderate rate on many compounds over those that grow very quickly on only a few. Genetic Structure of Pseudomonas
The Pseudomonas genophore is composed of a chromosome and perhaps any num ber of a varied collection of plasmids. The chromosome has been sized variously as 4 X 109 daltons in P. jiuorescens, P. stutzeri, and P. oleovorans and 7 X 109 daltons in P. aeruginosa by renaturation kinetic measurements (I), and as 2.4 X 109 daItons (in P. aeruginosa) by a combination of physical methods (J. M. Pemberton, personal communication). This discrepancy is hard to reconcile because there is no obvious reason to fault either investigation. One rather unlikely, but nevertheless tantalizing, possibility is that Pseudomonas has multiple chromosomes. More likelY, either there is a flaw in one of the determinations or Pseudomonas is very heterogeneous in terms of its DNA content (no strain was common to both investigations). Based on analogy to other prokaryotes, the 2.4 X 109 dalton value appears to be more proba ble. At least some strains of Pseudomonas harbor one or more plasmids in addition to the chromosome. These include fertility factors and R factors (see 36 for a review), a mercury resistance factor (15), plasmids coding catabolic enzymes (dis cussed below), and cryptic plasmids (32, 46, 56). One report indicates that P. aeruginosa strains gradually lose plasmids after isolation and upon laboratory cul ture (32). If, as suggested below, genetic transfer occurs in natural environments at a frequency sufficient to have had a -decided genetic effect upon evolution in the genus, it seems reasonable to consider the genetic structure of the population. As with eukaryotic populations, the individual is the direct target of selective forces, but the long-term effect mediating evolutionary change is variation in the genetic structure of the population; crudely, this means variation of allele frequencies. The Pseudomo nas genophore can be regarded as consisting of three classes of genes: (a) essential, (b) often useful, and (c) occasionally useful (59). Genes of the first two sets are most likely all located on the chromosome. Genes in the last set, including those for catabolic pathways, may be located on the chromosome of one strain but be plasmid borne or absent in others. Continual interstrain transfer of these plasmids assures a constant reassortment of nutritional types; transfer of chromosomal loci insures that combinations not achievable by plasmid transfer will also arise. PLASMIDS Defective Phage
The first reliable transductional system in P. pUlida was described by Gunsalus and his co-workers (16), utilizing two wild-type strains. One of these, now called PRS I, was isolated as A.3.12 by Stanier from a lactate enrichment and is the strain used
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in his laboratory for extensive studies of the biochemistry and regulation of aromatic acid catabolism. It has been designated the type strain of P. putida (70). The other was isolated in Gunsalus's laboratory from a camphor enrichment as strain C l. This strain is now designated PpG1 (or PpG2, a colony morphology mutant of PpG 1), but was for a short while termed PUGI (or 2). The phage employed was pf16 or its host-range mutant, pf16.h2. The latter is capable of plating on either PRSI or PpG2, and like the wild type is capable of mediating generalized transduction. Initial studies (l6) indicated that interstrain transduction between PRSI and PpG2 can be accomplished. The selection was for growth of PpG2 with mandelate, a compound normally unable to support the growth of this strain (Figure I). Both strains are capable of growth with benzoate; but PpG2 is unable to utilize D- or L-mandelate or benzoylformate. Chakrabarty et al (16) speculated that this is because of a complete lack of the genes involved. This contention is supported by D{-)mandE!late
! ! !
L{+)mandelate
benzoylfo"mate
be
nz
aldehyde
!
benzoalle
p-hydroxybenzoate
+ .. l+
catechol
co
protoC
!
'Y-carboxymuconolactone
(+) muconolactone
MI(catCl
J
Chuate
/l-carboxy-c,s,c,s-muconate
C/s,c,s muconate MLE (catB)
+
-----...
�
/l-ketoadipate enol lactone
!3
- , ,
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/l-ketoadipyl-CoA
+
acetYl-Co A + s u cci nate
Figure 1
The {3-ketoadipate pathway for aromatic acid dissimilation. Gene and enzyme
designations are as used in the text. Bold arrows indicate enzymes common to PRS I and PpG2, light arrows those of PRSI only.
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PSEUDOMONAS
509
the following findings: (a) no activity for any of the four assayed enzymes can be found in PpG2; (b) there is no material in PpG2 that gives an immunological cross-reaction with antibodies toward two PRSI mandelate enzymes; and (c) when the donor strain is deficient in mandelate dehydrogenase, so are all PpG2 mdl+ transducants (i.e. PpG2 cannot complement an mdlB mutation). Further investigation of this system (17) established that it is unusual in that the mechanism of establishment of mdl+ PpG2 strains appears to be via lysogenization by a defective phage particle carrying the mandelate genes. The evidence underlying this assertion can be summarized as follows: (a) the buoyant density of most mdl carrying transducing particles is intermediate between that of the pfu peak and the peak of met-carrying particles, a result consistent with their carrying a mixture of phage and bacterial DNA (which differ substantially in buoyant density). The met carrying particles are assumed to contain only bacterial DNA; (b) upon UV irradia tion and superinfection, lysates are formed that transduce mdl genes at elevated frequency; (c) many of the transductants are immune to vegetative superinfection by pf l6.h2; (d) the mdl genes segregate at a high spontaneous frequency (about 10-3 per bacterial division), which can be increased by UV irradiation; and (e) mdl+ PpG2 transductants show a satellite band of DNA on CsCI gradients inter mediate between the band of the main bacterial DNA and that of pfl6.h2 DNA; the satellite band is not shown by wild-type PpG2 or cured transductants. The evidence thus seems compelling that the mandelate+ phenotype in these transduc tants is a consequence of their acquisition of a (presumably extrachromosomal) element composed of (presumably covalently linked) phage and bacteria DNA. Calculations indicate that the transducing particles probably contain 35-70% bac terial DNA. There is conflicting evidence concerning the number of copies of this element (termed pfdm) per cell. Initial assays of the level of enzyme activity (16) suggest that there is one copy per chromosome, since they are about equal in the transduc tants and in the donor. Later studies (17), however, give 3-6-fold higher levels of activity in the transductants than in PRS1, suggesting the possibility of multiple copies. I have projected Figure 5 of Chakrabarty & Gunsalus (17), determined the relative areas of chromosomal and satellite peaks, and concluded that pfdm com prises 3.9% of the DNA of these transductants. If we assume a mol wt for the transducing fragment equal to that of the wild-type phage (9 X 107 daltons) (J. F. Niblack, personal communication) and a mol wt of 2.4 X 109 for the chromosome, one copy of pfdm per chromosome should represent 3.8% of the DNA. Thus, it is likely that pfdm and the chromosome are present in equal numbers. Should the chromosome of P. putida be found to be much larger than the above value, we should conclude that a larger number of copies of pfdm are present. It should be noted that phage pfl6 is a virulent phage, apparently totally incapable of establishing lysogeny and capable only of generalized transduction. However, these findings indicate that it is able to recombine with the chromosome to produce transducing particles, which appear to be of the type mediating specialized transduc tion (51). Although generalized transducing particles in Salmonella phage P22 have been reported to contain some phage DNA [(65); but cf (29) for a conflicting view],
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there is no evidence that such particles can establish lysogeny. Phage Pi, however, seems to be quite similar to pfl6 in that PI dl particles seem to have subst,antially the same structure as pfdm and are selected in crosses in which the recipient lacks the region being transduced (45). Thus, lack of homology may select for transduc tants by repliconation (20) rather than recombination. Because there are a number of nutritional differences between the two strains involved here (70), such specula tion is open to test. Degradative Plasmids The other major class of plasmids considered in this review is that comprised of plasmids carrying genes that specify enzymes of dissimilatory pathways. Chakrab arty has suggested (II) that these be termed degradative plasmids, and that nomen clature is adopted here. Five pathways are claimed to be specified by genes with plasmid location in one or more strains of Pseudomonas: camphor, octane, naphtha lene, m-tolute, and salicylate. They are discussed individually. Rheinwald et al (58) have presented evidence that the path way via which D or L-camphor is converted to isobutyrate is coded by plasmid-borne genes. The evidence rests on segregation frequencies and upon the self-transmissible nature of the cam genes. There are, however, conflicting reports concerning trans ductional linkage of these genes. Rheinwald et al have presented evidence indicating all the genes involved in the conversion of camphor to isobutyrate that were tested are cotransducible at high frequency. Thus, camA, camB, and camC (which code the three subunits of the first enzyme of the pathway) are closely linked to camD (second enzyme) and to camG (one of three subunits of the third enzyme). Several other markers, not further (:haracterized biochemically, that render the cell unable to utilize camphor but still able to ,use isobutyrate were shown also to cotransduce with camABCDG. The authors assert that none of these genes cotransduces with genes that prevent the utilization of isobutyrate. For this and other reasons (outlined below), Rheinwald et al propose that the cam genes are located on a linkage group separate from that (presumably the chromosome) occupied by the genes involved in isobutyrate catabolism (ibu) . However, an earlier report (16) asserted that not only did cam-l (hydroxylase negative, therefore probably a comA, -B, or -C muta tion) cotransduce at high frequency with cam-2 (biochemically uncharacterized), but that both of these gene!; cotransduced with /pa-I. The latter marker is lihked to some genes governing tryptophan biosynthesis and is almost certainly chromoso mal. A possible explanation for this discrepancy is suggested below. Despite the conflicting linkage data, the evidence supporting an autonomous state for the cam genes appears good. First, the spontaneous rate of mutation to cam is high (about 10"-4 per cell division) and can be enhanced by mitomycin C at sublethal concentrations (to close to one per cell division). These cam mutants do not revert at detectable frequencies; they lack all assayed enzymes of the camphor pathway and fail to yield cam+ transductants when used as either donors or recipi ents. This suggests that these genes are resident on a plasmid too large to be transduced by pfl6 in its entirety (i.e. probably >9 X 107 daltons). Second, the THE CAM PLASMID
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PSEUDOMONAS
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ability to utilize camphor can be transferred either to a spontaneous cam- mutant of PpG1 or to other strains of Pseudomonas-including several P. aeruginosa strains and one P. fluorescens strain-by mixing donor and recipient cultures. Third, the CAM plasmid is incompatible with other known plasmids, including one containing part (at least) of pfdm plus additional chromosomal material (58) and the plasmid (OCT) carrying genes for octane dissimilation (14). Chou et al (19) have isolated heat-sensitive mutants that apparently fail to repli cate CAM at the restrictive temperature. The mutation is chromosomal and does not affect the replication of OCT. Loss of CAM does not commence until about eight generations after temperature shift, which implies either that there are multiple copies of CAM present (unlikely, as several hundreds would be required) or that the block affects the synthesis of a cellular component present in excess. Pseudomonas pu tida dissimilates octane via octanol, oc tanaldehyde, and octanoic acid (2). The octanoate is presumably further metabol ized by f3-oxidation. At least two inductive events are involved in allowing growth on octane, as octanol does not induce the octane monooxygenase, yet does serve as a carbon and energy source. The monooxygenase is composed of three proteins (57), and as the octanol and octanaldehyde dehydrogenases are distinct (2), at least five structural genes are involved in the conversion of octanol to octanoate. The evidence summarized below indicates that at least some of these genes reside on an extra chromosomal element. The introduction of the CAM plasmid from Cl strains into the strain used by Baptist et al (2) [called P. oleovorans (2, 14, 57), but actually probably P. putida (70)] can be accomplished at low frequency by conjugation (see above), but the exconju gants become unable to grow with octane (14). Most (>90%) are also unable to grow with octano!' If octanol-negative exconjugants are subsequently cured of the CAM plasmid by mitomycin C treatment (see below) or are selected for spontaneous octanol positive strains, the CAM plasmid is lost and the cells simultaneously become capable of growth on octano!' They remain octane negative and do not revert. It thus appears that at least part of the octane hydroxylase is coded by a plasmid designated OCT that is incompatible with CAM, and that the chromosome carries the octanol and octanaldehyde dehydrogenases. The failure of most CAM+ OCT- exconjugants to grow with octanol was attributed (14) to repression by the CAM plasmid of the expression of the chromosomal dehydrogenase genes. The fact that some of these exconjugants are octanol positive has not been satisfactorily explained. In addition to being incompatible with CAM, OCT is defined as a plasmid on the basis of its curability and transmissibility. The rate of curing of OCT in the presence of sublethal mitomycin concentrations goes up to about 7% from ,
caICBR
,
mdlC
!
omiER
�
ben
/
P. pulido
Figure 2
Comparison of gene arrangement in P. pulida and P. aeruginosa. Breaks indicate
lack of demonstrated linkage. For clarity, the fragments of the P. aeruginosa map are arranged to coincide with
the P.
pUlida map; such coincidence has
not
been experimentally demon
strated. Gene designations: ben, mdl, cat, pob, ant, and pea for the J3-ketoadipate pathway
42, 61, 62, 74-); pal for phenylalanine (74); pac for phenylacetate (74); (43, 74); ami for amidase (6); and net for nicotinate (44).
(see Figure 1; and for histidine
hut
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517
quite differently in the two species (60). Gene arrangement in the pseudomonads, as in the enterics (64, 72), seems to be a fairly stable property. This region represents a minimum of 14 operons in P. putida (44). The end-to-end span is 9% of the bacterial chromosome [based on distances derived from pf16. h2-mediated transductions, analyzed by the method of Wu (78), and assuming a transducing fragment length of 9 X 107 daltons, with a chromosome of 2.4 X 109 daltons]. This P. putida map includes every mutation blocking a catabolic pathway that we have mapped; thus, to the limits of present resolution, every chromosomal gene with catabolic function is located in this region. Such clustering cannot be random (52). At present, it is unknown whether there are genes with noncatabolic function interspersed among the genes shown. There is room for at least 300 addi tional genes between ben and pobA, and it would be extremely interesting to know what functions they specify. Our failure to isolate large deletions in this area (M. L. Wheelis and B. J. Leidigh, unpublished observations) suggests that there are essential functions coded between the identified catabolic genes. I thus surmise, without any firm experimental support, that in Pseudomonas genes with biosyn thetic and other "essential" functions are randomly distributed over the chromo some (i.e. they are not excluded from the region under discussion) but are virtually absent from extrachromosomal linkage groups, whereas genes with catabolic func tion have a decidedly nonrandom organization on the chromosome and are fre quently found on other linkage groups. REGULATION OF GENE EXPRESSION
Regulation at the genetic level has been studied in detail in only three systems in Pseudomonas: the hur, carBe, and ami loci. Of these, one appears to be negatively controlled and one appears to be under positive control; the regulation of the third system is unclear at this time. The ami System
The regulation and genetics of the aliphatic amidase of P. aeruginosa have been studied by Clarke and her co-workers. This inducible enzyme converts acetamide, glycollamide, or propionamide to corresponding carboxylic acids. Selection for use of formam ide (a weak inducer and poor substrate) as sole source of nitrogen has allowed the ready isolation of constitutive mutants. In addition, the selection from parent strains constitutive for amidase production of strains able to use higher amides (butyra,mide, bon source led 'to an elegant series of studies of experimental evolution (5, 8, 9). Transductional mapping of the genes involved has indicated very tight linkage of amiR (the regulator gene) and amiE (the amidase structural gene) (6), and amiR has been used as an outside marker for the ordering by three factor crosses of a number of amiE lesions, including some that involve changes in the substrate specificity of the enzyme (3). The ami loci appear to be transductionally unlinked to the (presumably structural) genes, mutation in which abolishes synthesis of isocitrate lyase, malate synthase, citrate synthase, or acetic thiokinase (68).
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All of the published data is compatible with amiE being subject to classical negative control by a repressor protein, the structural gene for which is amiR. A recent abstract (30), however, suggests the possibility of positive control, based on (a) the lack of any temperature-sensitive constitutives (i.e. inducible at low tempera ture, constitutive at high temperature) and (b) the isolation of two strains that are constitutive at low temperature and unable to synthesize amidase at high tempera ture, and whose amidase appears identical to wild type by several criteria. Although neither of these considerations is in direct conflict with the classical model for negative control, they are somewhat more consistent with the system's being subject to positive control. This is especially true of b, which would, in a negative control model, demand that a single mutation abolish the function of the inducer-binding site on the repressor protein (at least at high temperature) and render operator binding cold sensitive. While there is nothing intrinsically improbable about such a possibility, it is a mutant type not previously described, even in the lac system of Escherichia. It thus seems prudent to await the results of further experimentation before drawing conclusions as to the nature of the control of the P. aeruginosa aliphatic amidase. The hut System
Histidine is dissimilated by P. putida via a five-step pathway (71), the first four enzymes of which are specified by the genes hutHUIF. A study of the control of this system (43) indicated that although the detailed biochemistry differs from that in the enterics, the regulation shows some striking similarities. First, the inducer of all four enzymes is the same (urocanic acid, the first intermediate). Second, the system appears to be under negative control exerted by the product of hutC, which is located in the midst of thl� structural genes of the pathway. Although the evidence is solid for the enteric bacteria (e.g. see 69), in P. putida the only real evidence for negative control is the infrequency of pleiotropic negative mutants (one out of about 40) and the ease with which constitutives were isolated in hutC. [The latter were selected by alternating between culture with histidine and glutamate, the product of the pathway, thereby selecting organisms that commenced growth without a lag. Such selection, first used by Cohen-Bazire & Jolit (23), should be of general utility when few inductive events occur between substrate and product.] Thus, although there is really no good evidence for classical negative control in the hut system, neither is there evidence basically inconsistent with the (probably at least two) hut operons being subject to such control; however, the superinducibility of both hutC mutants is curious. The catBe System
Although all parts of the I�-ketoadipate pathway have been studied in P. putida in great physiological detail (see 50 for a review), only one of the several presumed "operons" has been examined with any genetical detail (73, 77). This is the catBC system, which codes two f'nzymes involved with catechol dissimilation (Figure 1): muconate-lactonizing enzyme (MLE) and muconolactone isomerase (MI). The in ducer, cis,cis-muconate, is the substrate of the first of the two enzymes (MLE) and
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PSEUDOMONAS
519
also the inducer of catechol oxygenase (CO) (the enzyme that produces it), although CO induction is noncoordinate with that of MLE and MI (49). About a quarter of the negative mutants isolated for this system were pleiotropically negative, i.e. MLE-MI-. Deletion mapping (73) established that all of these pleiotropic-negative mutations clustered at one end of the catB gene. The two likely explanations are that either these represent mutations in a gene with positive regulatory control function or they are polar nonsense mutations in catB. Evidence against the latter is that mutagen-forced revertants of a small deletion with pleiotropic effects contain an MLE with identical immunological properties and rates of thermal denatura tion (77). The original deletion therefore probably did not enter catB. In addition, some of these revertants were weakly or strongly constitutive for MLE(catB), MI(catC), and CO(catA). A single mutation thus is sufficient to change the nonin ducible phenotype to constitutivity, which strongly suggests a regulatory gene (termed catR). This gene presumably produces a product that turns on the catBC "operon" and catA (presumably at separate sites). Two curious facts remain unex plained. First, why is CO constitutive in the constitutive revertants of catR-, yet normally inducible in all catR- strains? And second, why are no catR- mutants or revertants temperature sensitive [if the catR product were a protein, one would expect that many of the primary and secondary mutants would be temperature sensitive (e.g. 38)]? EVOLUTIONARY CONSIDeRATIONS OF GENETIC EXCHANGE
In addition to the discussion here, the interested reader is referred to the excellent discussions of the evolution of catabolic pathways in (4, 21, 35). It was fashionable for a time to consider that genetic exchange among bacteria was a laboratory artifact (e.g. 34) and of no consequence in natural populations. This view is now changing, and I discuss it with particular regard to Pseudomonas. The fact that pseudomonads are now recognized as carrying a variety of transmissible plasmids argues compellingly for the operation of transfer functions in nature, for we would expect such functions to have been lost if not selected. The possibility exists, of course, that what we term transfer functions have in reality other roles and that transfer is simply a side effect of these other functions; until there is some evidence for such pleiotropy, however, the simplest approach is to assume that the presence of self-transmissibility reflects the selection of such a phenotype. We con clude that genetic exchange, at least of plasmids, is important in natural populations. It should be noted that the spread of R factors among the enteric bacteria provides a striking and widely known example of the natural importance of plasmid transfer in another group of gram-negative bacteria, although one with a drastically different ecology. A similar conclusion about the importance of genetic transfer of chromosomal loci derives from consideration of the possible roles of chromosomal clustering. The degree to which genes with catabolic function are clustered, clearly in a supraoper onic fashion, defies explanation on the basis of shared elements of control of gene
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expression. We therefore proposed (74) that the selective advantage was to allow the simultaneous transfer of the genes involved. This argument for the separate operons of a single biochemical pathway is clear, because any one part of a pathway may be physiologically functionless without the rest. For separate pathways, the argu ment is less clear but can still be applied: transfer of a genetic region containing the determinants of more than one catabolic pathway would allow the recipient of this region to gain several new pathways in a single exchange event [see (44) for addi tional treatment of both this point and the role of duplication in evolution (discussed below)]. Alternatively, the clustering of genes involved in different catabolic path ways could have arisen from selection involving gene dosage effects. Thus, if the probability of gene transfer in wild populations is related to gene dosage, those genes whose transfer is most advantageous might come to be located in proximity to the origin of replication. Other effects of position relative to the initiation site for replication, such as raising (or lowering) the maximal level of gene expression, are less likely to have been selected because they are obtainable more simply and precisely by other means, such as variation in promoter specificity. This argument for the existence of genetic exchange among natural populations of bacteria says nothing about its actual frequency. It simply asserts that such exchange has been an important force in the evolution of the pseudomonads. In fact, because genetic exchange must be a second-order reaction at best and because natural Pseudomonas population densities are usually probably rather small (at least compared to densities of enterobacterial populations), the actual frequency of ex change must be rather low. A naive expectation would be that modification and restriction systems might be a liability because they could reduce this frequency to the level of nonexistence. In fact, at least as far as episome transfer is concerned (10), restriction does not seem to occur in strains of fluorescent pseudomonads, with the exception of P. aeruginosa. P. aeruginosa is a quite precisely definable and homoge neous species in terms of both phenotypic traits and DNA-DNA homology group ings, in contrast to the other species of pseudomonads, which are relatively heterogenous (53). This suggests that P. aeruginosa has been genetically insulated because of its restriction system, at least relative to other pseudomonads, among which there has been at least some interspecific exchange. Indeed amston (54) has argued from protein primary sequence data for relatively recent intergeneric genetic exchange (between Acinetobacter and Pseudomonas putida). Assuming, then, that genetic exchange does occur between bacteria in the wild at a rate sufficient to allow selection of various gene arrangements, it is possible to construct a model for the evolution of catabolic pathways that avoids a paradox in the Horowitz (37) "retroevolution" theory as applied to catabolic pathways. Horo witz postulated that the sequential steps in a biosynthetic pathway possess a filial relationship to each other, in that the genes coding the enzymes involved are derived one from another by duplication. Although this idea is fairly generally accepted for biosynthetic pathways, its application to the evolution of catabolic pathways has encountered some difficulties (e.g. see 35). Most of the objections (impermeability of intermediates, heterogeneity of the chemistry of sequential reaction steps, etc) seem to me to apply with equal force to both catabolic and anabolic pathways. There .
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is, however, one major difference between the two kinds of pathways, and that difference lies in the effect that the relative rates of duplication and recombinational loss of duplicated material will have (44). The rate of recombinational loss, in every system studied to date, is much higher than the rate of duplication; thus a duplicated region will be lost before there has been sufficient divergence between the two copies to both change the function of one and render the two dissimilar enough to lower the frequency of recombinational loss. The difference between biosynthetic and dissimilatory pathways with regard to this argument lies in the differences in inten sity and duration of selection that will be applied. Selection for a biosynthetic capability could quite easily have been continuous and strong, and in the systems studied to date, continuous selection can maintain a duplication indefinitely. The situation is different with respect to catabolic pathways, at least later in evolutionary history when the vast array of catabolic pathways characteristic of modern soil saprophytes presumably evolved. In this case, selection was almost certainly inter mittant, and the period between selective intervals was probably relatively long, especially for some of the more exotic pathways. Although the initial stages in the evolution of catabolic metabolism may have involved duplications (indeed the selective forces would probably have approached those guiding the emergence of anabolic capability), we have proposed (44) that later diversification of catabolic functions involved divergent evolution of strains, fol lowed by genetic exchange between them to yield recombinants that possessed the abilities of both parents. The end result is the same as in the case of duplication and divergence; the only difference being that genetic exchange allows the two diverging genomes to be protected against recombinational loss of duplicated regions because they are in different organisms. It should be noted that although I have accepted the likelihood of retroevolution both as the mechanism by which anabolic pathways evolved and as being possibly implicated in the early stages of development of catabolic pathways, there is no intellectual necessity for this. Gene exchange and the selective forces arising therefrom provide a formal alternative to retroevolution at all stages of biological evolution. One of the attractions of the retroevolution hypothesis is that it readily explains the genesis of operons, of which two steps can be envisaged: (a) the genes to be included must be brought into close proximity and (b) they must be subjected to common control. Presuming retroevolution involved principally tandem duplica tions, the first requirement is automatically fulfilled. If we abandon the retroevolu tion hypothesis for catabolic pathways, we must assume that the genes of an evolving pathway would be scattered on the chromosome. As we have seen, genetic transfer can provide a selective pressure for the clustering of genes with related function independently of shared control elements and may thus have been instru mental in forcing gene arrangements that later permitted the formation of operons (74). The same argument can, of course, be applied to the generation of plasm ids. Most dissimilatory plasmids probably could not have grown by gradual accretion of genes from the bottom up, because of the rarity, instability, or inability to cross the cell membrane of biochemical intermediates. Thus, acquisition of plasm ids probably
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confers no selective advant2lge unless they carry most or all of the determinants of a pathway. Therefore, on th,! assumption that most of such plasmid-borne pathways are ultimately derived from chromosomal genes, it seems likely that chromosomal clustering is a necessary intermediate in the genesis of dissimilatory plasmids. If such plasmids are indeed derived by recombinational excision from the chromo some, at least some of those more recently formed may in fact be able to reintegrate with the chromosome. Degradative plasmids may thus be in equilibrium between autonomous and integrated states. Such an equilibrium could explain the anomalous linkage results obtained for the CAM plasmid (see above) and is also consistent with the strain differences with regard to the genes for naphthalene dissimilation (plas mid-borne or chromosomal, depending upon strain) and with the ability of CAM to mobilize the chromosome. CONCLUDING REMARKS
Genes that specify enzymes with catabolic function in Pseudomonas appear in one of two distinctive linkage modes: they either are found in a small region of the chromosome or else are borne on plasmids. I interpret both kinds of topology as having been selected for their facilitation of genetic transfer. This sytem is of considerable interest because it may assist materially in the elucidation of the importance of both genetic recombination in the evolutionary history ofprokaryotes and the genetical structure of microbial populations in the wild. In my view, genetic exchange events play a crucial role in determining the genetic structure of such populations over both short and long time periods. In the short run, the spread of infectious plasmids can determine the extent to and rapidity with which a microbial population can degrade complex organic compounds; over longer periods of time, the evolution of novel abilities may depend upon recombination events. From a practical standpoint, an operative genetic approach to catabolism in a nutritionally versatile group of soil saprophytes may be immeasurably valuable in the construc tion of microbial strains capable of degrading some of the synthetic and noxious chemicals that man inflicts on the biosphere in increasing amounts. Even if such artificial strain construction proves not to be a feasible solution, the understanding of microbial population dynamics in nature is essential to sane environmental policy determination. Literature Cited 1.
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69:40-47 3. Betz, J. L., Brown, J. E., Clarke, P. H., Day, M. 1 975. Genet. Res. 23:335-59 4. Betz, J. L., Brown, P. R., Smyth, M. J., Clarke, P. H. 1 974. Nature 347:26 1 -64 5. Betz, J. L., Clarke, P. H. 1 972. J. Gen. Microbial. 73 : 1 6 1-74
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