JOURNAL OF BACTERIOLOGY, July, 1966Copyright © 1966 American Society for Microbiology

Vol. 92, No. IPrinted in U.S.A.

Suppression of Fluorophenylalanine Resistance byMutation to Streptomycin Resistance in

Pseudomonas aeruginosaJUDITH A. WALTHO' AND B. W. HOLLOWAY

School of Microbiology, University of Melbourne, Victoria, Australia

Received for publication 16 February 1966

ABSTRACTWALTHO, JUDITH A. (University of Melbourne, Victoria, Australia), AND B. W.

HOLLOWAY. Suppression of fluorophenylalanine resistance by mutation to strepto-mycin resistance in Pseudomonas aeruginosa. J. Bacteriol. 92:35-42. 1966.-Fluorophenylalanine-resistant mutants (fpa-r) of Pseudomonas aeruginosa havebeen isolated. By cotransduction analysis, the mutations were shown to have atleast two chromosomal locations. One locus (fpaA) showed linkage to three othermarkers, str, try-3bi, and arg-3, and the order of these four linked markers wasfound to be try-3bi, arg-3, fpaA, str. The linkage relationships of the other fpa lociare not yet known. The phenotypic expression of resistance at the fpaA locus canbe suppressed by mutation of the str locus from str-s to str-r, whereas that at anunlinked fpa locus cannot.

Our earlier finding (9) that related structuralgenes in Pseudomonas aeruginosa rarely show theclustering, characteristic of Escherichia coli andSalmonella typhimurium (for review, see 1), hasencouraged us to learn more of the controlmechanisms in biosynthesis in P. aeruginosa.As part of this project, we have looked foranalogue-resistant mutants, as these have provedparticularly useful in the study of the geneticmechanism of repression in E. coli (20). Geneticstudies on mutants of P. aeruginosa resistant toone such analogue, DL-p-fluorophenylalanine,form the substance of this paper; biochemicalstudies on the mutants will be published sepa-rately.

MATERIALS AND METHODS

Media. Nutrient broth (NB): Difco nutrient broth(0.8%) plus Difco yeast extract (0.5%). Nutrient agar(NA): beef extract (Bonox; 0.1%), peptone (Evans;1%), sodium chloride (0.8%), agar (Davis; 1.2%).Layer agar (LA) for phage assays: NB plus 1% Difcoagar. TNM buffer: tris(hydroxymethyl)aminometh-ane (Tris; 0.01 M), NaCl (0.15 M), MgSO4 (0.01 M,pH 7.4). Minimal medium (MM): that of Vogel andBonner (26), solidified where necessary with 1.5%Difco agar.

General cultural procedures. The procedures werethe same as those used previously (13, 16).

Strains. All mutants were derived from P. aerugi-nosa strain 2 (ATCC 15693). The various auxotrophsfor mapping experiments were previously described(9, 15) and are listed in Table 1. The bacteriophagestrain used was F116 (14).

Isolation of streptomycin-resistant mutants. Ap-proximately 1010 organisms were plated onto NAcontaining 250 jug/ml of streptomycin (Glaxo), andthe plates were incubated at 37 C. Resultant colonieswere streaked on NA; from these streaks, singlecolonies were cultured and subsequently checked forhomogeneity of resistance. Scoring of transductantsfor streptomycin resistance or sensitivity was doneby patching colonies onto NA containing 250 ,ug/mlof streptomycin.

Isolation offpa-r mutants. An overnight culture ofstrain 2 in NB was diluted to give approximatelyfive bacteria per milliliter; 1-ml portions of this wereused to inoculate fifty 10-ml amounts of fresh broth.These were incubated, with shaking, overnight. Thosein which growth occurred (40/50) were centrifuged,and the cells were suspended in buffer to give ap-proximately 4 X 109 viable cells per milliliter. A 0.1-mlsample from each of these suspensions was spreadonto a plate of MM containing 1 mg of fluorophenyl-alanine (FPA)/ml. After incubation for 3 to 4 daysat 37 C, colonies appeared on almost all the plates(at a frequency of about 5 X 10-8 inoculated cells).Two colonial types were observed: a cream-colored,entire-edged, butyrous type, and a brown, entire-edged, viscid type.

1 On leave from Commonwealth Scientific & Indus-trial Research Organization, Division of Food Preser-vation, Ryde, New South Wales, Australia.

35

WALTHO AND HOLLOWAY

TABLE 1. Bacterial strains used in this study; mutants were derived from strain 2 except where stateda

Strain no. Description Strain no. Description

PrototrophicPrototrophic str-rmet-2a str-rbmet-Imet-IC str-rbmet-2bcys-lcys-2 str-rbhom-ahom-barg-J

arg-2arg-3lys str-rbilva-lilva-2 str-rb

pro-I

pro-2 str-rbpro-3 str-Hrtry-]try-3bi str-rbtry-3bii str-rbtry-2ura-l str-rbura-2 str-rbura-3thr str-rbhis-]his-2his-3 str-rbgly or ser, str-rbgly str-rbser str-rbleu-2 str-rbleu-l str-rbade-l str-rbade-2 str-rbade-3try-3bi

2-2001, 2-2004, 2-2006,2-2007, 2-2008, 2-2009,2-2011, 2-2012, 2-2013,2-2014, 2-2015, 2-2015P,2-2016P, 2-2017P,2-2018P, 2-2019P,2-2020P, 2-2021P,2-2022P, 2-2023P,2-2025P, 2-2026P,2-2027P, 2-2028P,2-2029P, 2-2030

2-2005, 2-2014P?\2-2018, 2-2024 J

2-2040

2-2041, 2-2042, 2-2043}

2-2458-1, 2-2458-32-24584, 2-2458-52-2458-6, 2-2458-72-2458-9, 2-2458-11J

2-2458-2

2-2179-1, 2-2179-2

2-2004-1

2-1019, 2-1020, 2-10212-1023, 2-1030, 2-10462-1048, 2-1049, 2-10502-1051, 2-1052, 2-10532-1054, 2-1055, 2-1056,

2-1057

Prototrophic, fpa-r derived from 2Group A (see Table 2)

Prototrophic, fpa-r derivedGroup B (see Table 2)

from 2

Prototrophic, str-r derived from 2

Prototrophic, fpa-r and str-r, derivedfrom 2-2040

try-3bi, fpa-r, and str-r; derived from2458. Group C (see Table 2)

try-3bi, fpa-r, and str-r; derived from2-458

try-3bi, fpa-r; derived from 2-179

Prototroph, str-r, suppressed FPA-resistance phenotype derived from2-2004

phe

a Strains are str-s and fpa-s except where noted as otherwise. Strain 2-179 was described by Hollowayet al. (15). Strains listed above 2-179 (left) were described by Fargie and Holloway (9). Strains below2-179 are described in this paper.

I Derived from 2S.

Colonies taken from these plates were streaked onNA, and a single colony from each streak was culturedand subsequently checked for homogeneity of re-sistance to FPA. By this procedure the isolation ofsibling mutants was avoided. Following previouspractice, the individual isolates were given isolatenumbers; the letter P following the number indicatesthe latter of the two colonial types described above.No other difference in behavior has yet been foundbetween these two types of mutants.

Mapping FPA resistance. As an adequate sexualmating system is not yet available for mapping in P.aeruginosa strain 2, mapping was carried out bycotransduction techniques. With bacteriophage F116(9), 36 different auxotrophic loci, identified from

earlier work and listed in Table 1, together with a

newly isolated series of aromatic auxotrophs, wereselected, and the linkage relationships of the FPA-resistant mutants to these loci were tested by thefollowing method.

Cells (4 X lO8) of each auxotrophic mutant, sus-pended in TNM buffer, were spread on MM. PhageFl 16 was propagated in turn on each of the inde-pendently isolated FPA-resistant prototrophs, anda loopful (about 0.05 ml) of each of these phagepreparations was then spotted onto each of the auxo-troph-spread plates, which were then incubated at37 C. After 2 days of incubation, several hundredprototrophic transductants appeared in each area ofadded phage. Each group of transduced colonies

22S2-4102-3602-4662-3012-3412-4302-3662-3862-3032-3802-7712-5492-3082-4162-612-5462-6942-3222-4582-4472-3302-6312-6352-7672-5642-3872-3062-4802-4512-6252-624240924852-5862-5002-3712-179

1-

36 J. BACTERIOL.

SUPPRESSION OF ANALOGUE RESISTANCE

was sampled with an inoculating loop, and the massof cells was smeared onto MM containing FPA. Ifcotransduction had occurred between the fpa locusand the prototrophic alleles of any of the auxotrophicmarkers, some of the colonies would have becomephenotypically resistant to FPA. For those combina-tions where any growth appeared on MM plus FPA,a quantitative transduction test (16) was then carriedout by use of the appropriate phage preparation andauxotroph.

Metabolite analogues. Metabolite analogues wereobtained from the Mann Fine Chemicals, Inc., ex-cept where stated otherwise. The 1,2, 4-triazole-3-alanine was the generous gift of E. Lilly & Co.

RESULTS

Sensitivity of P. aeruginosa to metaboliteanalogues. The sensitivity of P. aeruginosa strain2 to a variety of metabolite analogues wasmeasured in the first instance by placing a fewcrystals of the analogue onto the surface of anMM plate seeded with 4 X 108 viable organisms.The plates were then incubated for 2 days at37 C. The following antimetabolites showed nosignificant inhibition of growth (compounds inparenthesis are the corresponding naturalmetabolites): L-canavanine (arginine), allylglycine (cysteine), 5 - methyl - DL - tryptophan(tryptophan), L-ethionine (methionine), 2-amino-4-methyl-pyrimidine (pyrimidines), barbituricacid (uracil), 2-chloro-4-aminobenzoic acid(p-aminobenzoic acid), oxythiamine hydro-chloride (thiamine), DL-desthiobiotin (biotin),DL-methionine sulfoxide (glutamic acid), DL-pantoyl lactone-Na salt (pantothenic acid),3-2-thienylalanine (phenylalanine), benzimida-

zole (adenine, guanine), D-ethionine (methionine),2, 6-diamino purine hemisulfate (adenine), 1,2,4-triazole-3-alanine (histidine), and 2-thiouracil(uracil). (The L-ethionine and D-ethionine werefrom Calbiochem; 2-thiouracil was from theSigma Chemical Co., St. Louis, Mo.)By contrast, DL-p-fluorophenylalanine (FPA)

gave a marked inhibition of bacterial growthunder these conditions. The inhibition by FPAwas measured quantitatively, and it was f3undthat a concentration of 1 mg/ml (5.4 X 10-3 Mof the DL form) was required to inhibit completelygrowth of P. aeruginosa 2. This inhibition couldbe reversed by the addition of phenylalanine butnot of tyrosine, tryptophan, indole plus serine,anthranilic acid, phenylpyruvic acid, p-hydroxy-benzoic acid, or p-aminobenzoic acid, indicatingthat the reason for this activity of FPA is almostcertainly due to its competition with phenylal-anine alone.

Genetic analysis of FPA resistance. A numberof independent FPA-resistant mutants were

isolated and their linkage relations determined.In the preliminary screening test, it was foundthat, with most of the FPA-resistant isolates, thelocus for resistance (fpa-r) showed linkage totwo auxotrophic loci, try-3bi and arg-3. Theremainder showed no detectable linkage to anyof the auxotrophic loci tested.The fpa-r loci of 30 FPA-resistant prototrophs,

of 3 FPA-resistant tryptophan auxotrophs, andof 3 FPA-resistant streptomycin-resistant proto-trophs were examined for cotransduction withtry-3bi and arg-3. In addition, cotransductionfrequencies for try-3bi and str, arg-3 and str, andarg-3 and try-3bi were measured.The linkage relations of these mutants are

shown in Table 2, where it can be seen that,excluding four of the FPA-resistant prototrophs,all FPA-resistant mutants gave comparablefrequencies of cotransduction with try-3bi andarg-3 and can be considered to have mutated atthe one locus. This site has been called fpaA,whereas the four unlinked mutations have beenreferred to by their allele numbers.

In addition, it can be seen that try-3bi, arg-3,and str are all able to be cotransduced. From thecotransduction frequencies, it can be deduced thattry-3bi is closer to arg-3 (52%) than it is to eitherfpaA (26%) or to str (7%), that arg-3 is closer tofpaA (60%) than is try-3bi (26%), that arg-3 iscloser to str (29%) than is try-3bi (7%), and thatarg-3 is closer to fpaA (60%) than it is to str(29%). These facts direct the unique order shownin Fig. 1.

Evidence confirming this gene order wasobtained by an examination of the crossoverclasses recovered in the various transductions.Two representative crosses are shown in Table 3,from which it can be seen that the low frequencyor absence of multiple crossover classes supportsthe order deduced from the cotransductionfrequencies given in Table 2.

Additional transduction tests confirmed thattry-3bii, try-i, and try-2, as distinct from try-3bi,show no linkage to fpaA, thus corroborating theprevious finding (9) that structural genes of thetryptophan pathway in P. aeruginosa are notclustered. Similarly, lack of linkage betweentry-3bii, try-i, try-2, and arg-3 was demonstrated.

Interaction of streptomycin resistance and FPAresistance. During the course of the linkageexperiments described above, it became necessaryto prepare stocks which were resistant to bothstreptomycin and FPA. Although this wasaccomplished readily in some cases, in others anunexpected reaction was obtained. For example,the FPA-resistant prototroph 2-2004 was platedon NA containing 250 ,ug/ml of streptomycin.

VOL. 92, 1966 37

WALTHO AND HOLLOWAY

TABLE 2. Cotransduction with markers try-3bi, arg-3, str, and FPA -resistant mutants

Phenotype No. ofDonor Recipient Selected Cotrans- b deter-

marker ductiona SE mina-Donor Recipient tions

try-3bi and FPA-resistant mutants

try-3bi-fpaA26 independent 2-458 try+ fpa-r str-s try fpa-s str-r try+ 26.2 1.5 51

isolates of groupA (see Table 1)

2-771 2-2458-4 try+ argfpa-s str-s try arg+ fpa-r str-r try+ 37.0 0 22 (prototroph) 2-2458-4 try+ fpa-s str-s try fpa-r str-r try+ 25.0 0 28 independent iso- 2-179 try+ fpa-r str-s try fpa-s str-s try+ 23.6 1.8 9

lates of group A(see Table 1)

2-2041, 2-2042, 2-179 try+ fpa-r str-r try fpa-s str-s try+ 26.3 4.6 32-2043

2 (prototroph) 2-2179-2 try+ fpa-s str-s try fpa-r str-s try+ 27.5 9.5 22-2041, 2-2042, 2-458 try+ fpa-r str-r try fpa-s str-r try+ 25.3 1.0 3

2-2043Grand mean 26.2 2.0

try-3bi and the fol-lowing FPA-re-sistant mutants

2-2005, 2-2014P, 2-458 try+ fpa-r str-s try fpa-s str-r try+ 0 0 102-2018, 2-2024P.

try-3bi-str9 independent iso- 2-458 try+ fpa-r str-s try fpa-s str-r try+ 6.0 0.6 10

lates of group A2 2-458 try+ fpa-s str-s try fpa-s str-r try+ 7.5 0 18 independent iso- 2-2458-2 try+ fpa-r str-s try fpa-r str-r try+ 4.0 0.8 8

lates of group A2-771 2-458 try+ arg fpa-s str-s try arg+ fpa-s str-r try+ 17.0 0 22-771 2-2458-4 try+ arg fpa-s str-s try arg+ fpa-r str-r try+ 6.0 0 22-2040 2-179 try+ fpa-s str-r try fpa-s str-s try+ 5.8 1.7 22-2041, 2-2042, 2-179 try+ fpa-r str-r try fpa-s str-s try+ 9.0 1.5 3

2-20432-2041, 2-2042, 2-2179-1 try+ fpa-r str-r try fpa-r str-s try+ 8.0 1.6 5

2-2043 2-2179-2Grand mean 6.8 1.0

arg-3 and FPA-re-sistant mutants

arg-3-fpaA26 independent 2-771 arg+ fpa-r str-s arg fpa-s str-s arg+ 61.6 1.6 48

isolates of groupA

2-2041, 2-2042, 2-771 arg+ fpa-r str-r arg fpa-s str-s arg+ 62.0 1.2 32-2043

2-2458-1, 2-2458-4 2-771 try arg+ fpa-r str-r try+ arg fpa-s str-s arg+ 46.8 4.4 62-2458-5, 2-2458-6

Grand mean 60.1 2.0

arg-3 and the follow-ing FPA-resist-ant mutants

2-2005, 2-2014P, 2-771 arg+ fpa-r str-s arg fpa-s str-s arg+ 0 0 82-2018, 2-2024P

38 J. BACTERIOL.

SUPPRESSION OF ANALOGUE RESISTANCE

TABLE 2. Continued

Phenotypes No. ofDonoRecpien ____________________ ____________________Selected Cotrans- Eb deter-Donor Recipient marker ductions s mina-

Donor Recipient tions

arg-3-str7 independent iso- 2-771 try arg+ fpa-r str-r try+ arg fpa-s str-s arg+ 28.7 3.2 9

lates of group C2-2041, 2-2042, 2-771 try+ arg+ fpa-r str-r try+ arg fpa-s str-s arg+ 30.3 0.9 3

2-2043Grand mean 29.1 2.9

arg-3-try-3bi2-771 2-458 try+ arg fpa-s str-s try arg+ fpa-s str-r try+ 47 0 22-771 2-2458-4 try+ arg fpa-s str-s try arg+ fpa-r str-r try+ 49 0 28 independent iso- 2-771 try arg+ fpa-r str-r try+ arg fpa-s str-s arg+ 54.1 3.3 10

lates of group CGrand mean 52.3 3.0

a Cotransduction frequencies were obtained by averaging the results obtained from the donor strainsgiven in the left-hand column.

b Calculated from Bessels formula for the standard deviation of means.

try-3bi arr-3

7

FIG. 1. Linkage relationships of try-3bi, arg-3,fpaA, and str. Figures represent percentage cotrans-duction.

Colonies appeared spontaneously after 48 hr ofincubation at a frequency of 10-'0/plated viablecells. After one such colony had been purified bystreaking on NA and a single colony had beenisolated and grown, it was found to be pheno-typically FPA-sensitive and streptomycin-re-sistant. About 50% of the colonies arising fromthe plating of 2-2004 on NA plus streptomycinshowed this loss of resistance to FPA. A number

of independently arising mutants with this char-acteristic were streaked on NA through severalsingle-colony isolations and were found to bestable and homogeneous. This loss of FPAresistance was not reversed by the presence ofstreptomycin. The change in phenotypic responseto FPA was also shown to occur in one directiononly, from the resistant to the sensitive state.When an FPA-sensitive organism mutates tostreptomycin resistance, no changes to an FPA-resistant phenotype are observed. All the colonieswhich had lost FPA resistance in this way grewon both NA and MM, thus indicating absenceof both auxotrophy and streptomycin dependence.Similar results were obtained with other strainscarrying the fpaA-resistant allele, and the effectis not limited to strain 2-2004.

It was also found that, when selection wasmade in this way for streptomycin-resistantmutants in some FPA-resistant strains, not onlydid this loss of FPA-resistance phenotype occur,but also 5 to 15% of the colonies appearing on

NA plus 250 ,ug/ml of streptomycin lost theirstreptomycin resistance during subsequent sub-culture on streptomycin-free media. We have no

TABLE 3. Recovery of recombinant classes in transduction experiments

Recombinant classaDonor Recipient Selection

fpa-r str-r fpa-r str-s fpa-s str-r fpa-s str-s

2-2041 (arg+ fpa-r str-r) 2-771 (argfpa-s str-s) arg+ 49 62 5 712-2001 (try+ fpa-r str-s) 2-458 (try fpa-s str-r) try+ 26 7 112 0

a Number of transductants isolated.

39VOL. 92, 1966

f-oa-A

WALTHO AND HOLLOWAY

explanation at present for this result, and it isbeing investigated in more detail.The above reactions have all been found with

the fpaA locus, which is linked to streptomycin.By contrast, when 2-2005, a strain in which theFPA-resistance gene is not linked to try-3bi,arg-3, or str, is plated on NA plus streptomycin,the colonies which arise as spontaneous muta-tions show a uniformly stable resistance to bothstreptomycin and FPA after streaking and sub-culturing on NA.The interaction, therefore, appears to be

concerned with the str locus and the fpaA locus,these loci being linked, and does not involve anyunlinked FPA-resistance loci.The most likely explanation of this effect is that

some streptomycin resistance mutations, but notall, can act as suppressors of this FPA resistancegene to which they are linked. This hypothesiscan be directly tested by taking a (presumably)suppressed strain, with the genotype fpaA-rstr-r (but phenotype FPA-sensitive, streptomycin-resistant), and separating by recombination thefpa and str genes. Thus, from the cross, fpaA-r(suppressed) str-r x fpaA-s str-s, recombinantswith the fpa-r phenotype should be recovered.Such a cross was carried out by use of 2-771(arg-3, fpaA-s, str-s) as the recipient in a trans-duction experiment with phage F116 propagatedon 2-2004-1 (arg-3+ fpaA-r (Su4) str-r) as donor.Selection was made for arg4 on MM, and theprototrophic transductants were individuallyscored for their response to FPA and strepto-mycin by patching onto MM plus FPA and NAplus streptomycin. A check on the phenotype ofthe actual broth culture used to prepare thetransducing phage in this experiment was madeby spreading about 109 washed cells over thesurface of an MM plus FPA plate. It was foundthat no colonies appeared after 48 hr of incuba-tion. Similar tests with 2-771 showed that itharbored no unexpected FPA-resistant clones(Table 4).

It is seen that, by selection for arg-3, it ispossible to segregate the str-r allele from thefpa-r allele and thus to remove the suppressor

TABLE 4. Appearance of FPA-resistant recombi-nants from a cross between the phenotypicallyFPA-sensitive parents, 2-2004-1 and 2-771,

with selection for arg-3+

No. of re- Phenotype of recombinantscombinants

tested fpa-s sir-s fpa-s slr-r fpa-r str-s fpa-r sir-r

236 81 78 78 0

effect. The frequencies of cotransduction de-scribed for the markers involved in this analysisare: arg-3-fpaA, 60%; arg-3-str, 29%. Itmight thus be expected that, provided no pref-erential crossing over occurs, the frequency ofcotransduction of arg-3 and fpaA, without theconcomitant cotransduction of str (a situationwhich would yield the unsuppressed and thereforephenotypically resistant fpa gene), would be 31 %.In fact, a value of 33 % was obtained. Our con-clusion, that a suppression of fpaA resistance bystr resistance occurs, is further corroborated bythe fact that, in the resulting recombinants inwhich the str-r allele is cotransduced with arg-3,the phenotype with respect to FPA is withoutexception the suppressed, i.e., the sensitive, form.

DIscussIoNIt has been shown that some, but not all,

spontaneous mutations to streptomycin resistancecan result in suppression of the phenotype of alinked gene directing FPA resistance. No suchsuppression action is imposed upon the FPA-resistance gene (or genes) which are unlinked tothe str gene.

This phenotypic interaction could lead toanomalous segregation data for the str and fpaAgenes. However, examination of the data ofTable 2, in those cases where both genes aresegregating, reveals no such anomalies. It isreasonable to assume that, fortuitously, none ofthe streptomycin resistance mutations used inthese cotransduction studies was of the sup-pressive type.The observation of suppression by some

mutations to streptomycin resistance is not new.In observing phenotypic repair of enzymedeficiency in E. coli by streptomycin, the existenceof a streptomycin-activated suppressor mecha-nism was suggested (12), while at the same timethe necessity for "competence" in the strepto-mycin-resistant mutants was stressed. Otherexamples of suppression by mutation to strepto-mycin resistance in E. coli have also been de-scribed (2, 3, 19).Our observation of str and fpaA interaction in

P. aeruginosa, however, is interesting because ofthe proximity of the two markers to each other.The mode of action of streptomycin and ofmutation to streptomycin resistance has been welldocumented for E. coli (4, 5, 7, 10, 11, 17, 21-25),and has led to the following concept. In a strepto-mycin-sensitive organism, streptomycin acts atthe level of the 30S ribosome subunit. It does notprevent attachment of the messenger ribonucleicacid (RNA) to the ribosomes, but rather inter-feres with the function of the complex so formed;

40 J. BACTERJOL.

SUPPRESSION OF ANALOGUE RESISTANCE

thus, the binding of specific soluble RNA typesto the complex is altered. This phenomenon thusgives rise to misdirection of amino acids intoprotein. The condition of streptomycin resistancewould appear to result from a mutation in thatpart of the chromosome determining ribosomalstructure, such that conformational changes arisewhich preclude the ability of streptomycin tointerfere with the function of the ribosome-messenger complex.The interaction between the function of the

closely linked genes for streptomycin resistanceand FPA resistance in P. aeruginosa lead us tosuggest that in this area of the chromosome weare dealing with a group of genes controllingribosome structure. The same proposal regardingthe str area of the E. coli chromosome has alsoappeared (6, 18). In the first-cited case, it wasfound that the gene for the ribosomal proteinspecific to strain K-12 is close to str; in thesecond, the gene for resistance to spectinomycin(which also acts at the 30S ribosome subunit) isclose to the str locus. In addition, it has beenshown with Bacillus subtilis that the genes forerythromycin resistance, and for the structure of16S, 23S, and 4S ribosomal RNA, are alsolocated close to str (8).To confirm our suggestion for P. aeruginosa,

it will be necessary to demonstrate firstly that theaction of streptomycin is the same in this organismas in E. coli, and secondly that FPA can act atthe level of translation.

ACKNOWLEDGMIENTS

This investigation was supported by a grant fromthe National Health and Medical Research Counciland a grant-in-aid from C.S.I.R.O. One of us(J. A. W.) was the recipient of a CommonwealthPost Graduate Award.

LITERATURE CITED

1. AMES, B. N., AND R. G. MARTIN. 1964. Bio-chemical aspects of genetics: the operon. Ann.Rev. Biochem. 33:235-258.

2. ANDERSON, W. F., L. GORINI, AND L. BRECKEN-RIDGE. 1965. Role of ribosomes in strepto-mycin-activated suppression. Proc. Natl. Acad.Sci. U.S. 54:1076-1083.

3. COUTURIER, M., L. DESMET, AND R. THOMAS.1964. High pleiotropy of streptomycin muta-tions in Escherichia coli. Biochem. Biophys.Res. Commun. 16:244-248.

4. Cox, E. C., J. R. WHITE, AND J. G. FLAKS. 1964.Streptomycin action and the ribosome. Proc.Natl. Acad. Sci. U.S. 51:703-709.

5. DAVIES, J. E. 1964. Studies on the ribosomes ofstreptomycin-sensitive and resistant strains ofEscherichia coli. Proc. Natl. Acad. Sci. U.S.51:659-664.

6. DAVIES, J., P. ANDERSON, AND B. D. DAVIS.1965. Inhibition of protein synthesis by spec-tinomycin. Science 149:1096-1098.

7. DAVES, J., W. GILBERT, AND L. GoRINI. 1964.Streptomycin, suppression and the code. Proc.Natl. Acad. Sci. U.S. 51:883-890.

8. DUBNAU, D., I. SMITH, AND J. MARMUR. 1965.Gene conservation in Bacillus species. II.Proc. Natl. Acad. Sci. U.S. 54:724-730.

9. FARGE, B., AND B. W. HOLLOWAY. 1965. Ab-sence of clustering of functionally relatedgenes in Pseudomonas aeruginosa. Genet. Res.(Cambridge) 6: 284-299.

10. FLAKS, J. G., E. C. Cox, M. L. WrITING, ANDJ. R. WHITE. 1962. Polypeptide synthesis withribosomes from streptomycin-resistant anddependent E. coli. Biochem. Biophys. Res.Commun. 7:390-393.

11. FRIEDMAN, S. M., AND I. B. WEINSTEIN. 1964.Lack of fidelity in the translation of syntheticpolyribonucleotides. Proc. Natl. Acad. Sci.U.S. 52:988-996.

12. GORINI, L., AND E. KATAJA. 1964. Phenotypicrepair by streptomycin of defective genotypesin E. coli. Proc. Natl. Acad. Sci. U.S. 51:487-493.

13. HOLLOWAY, B. W. 1955. Genetic recombinationin Pseudomonas aeruginosa. J. Gen. Microbiol.13:572-581.

14. HOLLOWAY, B. W., J. B. EGAN, AND M. MONK.1960. Lysogeny in Pseudomonas aeruginosa.Australian J. Exptl. Biol. 38:321-329.

15. HOLLOWAY, B. W., L. HODGINS, AND B. FARGIE.1963. Unlinked loci affecting related biosyn-thetic steps in Pseudomonas aeruginosa. Nature199:926-927.

16. HOLLOWAY, B. W., M. MONK, L. HODGINs, ANDB. FARGIE. 1962. Effects of radiation on trans-duction in Pseudomonas aeruginosa. Virology18:89-94.

17. KAJI, H., AND A. KAI. 1965. Specific binding ofsRNA to ribosomes: effect of streptomycin.Proc. Natl. Acad. Sci. U.S. 54:213-219.

18. LEBOY, P. S., E. C. Cox, AND J. G. FLAKS. 1964.The chromosomal site specifying a ribosomalprotein in Escherichia coli. Proc. Natl. Acad.Sci. U.S. 52:1367-1374.

19. LEDERBERG, E. M., L. CAVALLI-SFORZA, AND J.LEDERBERG. 1964. Interaction of streptomycinand a suppressor for the galactose fermentationin E. coli K12. Proc. Natl. Acad. Sci. U.S.51:678-682.

20. MAS, W. K. 1961. Studies on repression ofarginine biosynthesis in Escherichia coli. ColdSpring Harbor Symp. Quant. Biol. 26:183-191.

21. MAGER, J., M. BENEDICT, AND M. ARTMAN.1962. A common site of action for polyaminesand streptomycin. Biochim. Biophys. Acta62:202-204.

22. SPEYER, J. F., P. LENGYEL, AND C. BASILIO.1962. Ribosomal localization of streptomycinsensitivity. Proc. Natl. Acad. Sci. U.S. 48:684-686.

23. SpoTTS, C. R., AND R. Y. STANIER. 1961. Mecha-

VOL. 92, 1966 41

42 WALTHO AN

nism of streptomycin action on bacteria: a uni-tary hypothesis. Nature 192:633-637.

24. SZER, W., AND S. OCHOA. 1964. Complexingability and coding properties of syntheticpolynucleotides. J. Mol. Biol. 8:823-834.

25. VAN KNIPPENBERG, P. H., J. C. VAN RAVENS-WAAY CLAASEN, M. GRIJM-VOS, H. VELDSTRA,AND L. BOSCH. 1965. Stimulation and inhibi-

D) HOLLOWAY J. BACEERIOL.

tion of polypeptide synthesis by streptomycinin ribosomal systems of Escherichia coli, pro-grammed with various messengers. Biochim.Biophys. Acta 95:461-473.

26. VOGEL, H. J., AND D. M. BONNER. 1956. Acetyl-ornithinase of Escherichia coli: partial purifi-cation and some properties. J. Biol. Chem.218:97-106.