Use of polymerase chain reaction to identify a leucyl tRNA in Streptomyces coelicolor

Polymerase chain reaction (PCR) was used to amplify a fragment of DNA encoding a tRNA that recognizes the abundant CUC leucine codon from the chromosome of Streptomyces coelicolor. Sequence analysis of the gene, designated leuU, indicated that it codes for a tRNA 88 nucleotides in length that shares 75% identity with the Escherichia coli tRNA^Leu_CUC, while it shares only 65% identity with the only other sequenced leucyl tRNA from S. coelicolor, the bldA encoded tRNA^Leu_UUA. Accumulation of the leuU tRNA was examined by Northern blot analysis and shown to be present at constant levels throughout growth in contrast to the bldA-encoded tRNA which shows a temporal pattern of accumulation [Leskiw et al., 1993. J. Bacteriol., 175, 1995–2005].     

Proteomics analysis of global regulatory cascades involved in clavulanic acid production and morphological development in <Emphasis Type="Italic">Streptomyces clavuligerus</Emphasis>

The genus Streptomyces comprises bacteria that undergo a complex developmental life cycle and produce many metabolites of importance to industry and medicine. Streptomyces clavuligerus produces the β-lactamase inhibitor clavulanic acid, which is used in combination with β-lactam antibiotics to treat certain β-lactam resistant bacterial infections. Many aspects of how clavulanic acid production is globally regulated in S. clavuligerus still remains unknown. We conducted comparative proteomics analysis using the wild type strain of S. clavuligerus and two mutants (ΔbldA and ΔbldG), which are defective in global regulators and vary in their ability to produce clavulanic acid. Approximately 33.5 % of the predicted S. clavuligerus proteome was detected and 192 known or putative regulatory proteins showed statistically differential expression levels in pairwise comparisons. Interestingly, the expression of many proteins whose corresponding genes contain TTA codons (predicted to require the bldA tRNA for translation) was unaffected in the bldA mutant.     

Pleiotropic regulatory genes bldA,adpA and absB are implicated in production of phosphoglycolipid antibiotic moenomycin

Unlike the majority of actinomycete secondary metabolic pathways, the biosynthesis of peptidoglycan glycosyltransferase inhibitor moenomycin in Streptomyces ghanaensis does not involve any cluster-situated regulators (CSRs). This raises questions about the regulatory signals that initiate and sustain moenomycin production. We now show that three pleiotropic regulatory genes for Streptomyces morphogenesis and antibiotic production—bldA, adpA and absB—exert multi-layered control over moenomycin biosynthesis in native and heterologous producers. The bldA gene for tRNA^Leu_UAA is required for the translation of rare UUA codons within two key moenomycin biosynthetic genes (moe), moeO5 and moeE5. It also indirectly influences moenomycin production by controlling the translation of the UUA-containing adpA and, probably, other as-yet-unknown repressor gene(s). AdpA binds key moe promoters and activates them. Furthermore, AdpA interacts with the bldA promoter, thus impacting translation of bldA-dependent mRNAs—that of adpA and several moe genes. Both adpA expression and moenomycin production are increased in an absB-deficient background, most probably because AbsB normally limits adpA mRNA abundance through ribonucleolytic cleavage. Our work highlights an underappreciated strategy for secondary metabolism regulation, in which the interaction between structural genes and pleiotropic regulators is not mediated by CSRs. This strategy might be relevant for a growing number of CSR-free gene clusters unearthed during actinomycete genome mining.     

The Use of the Rare TTA Codon in Streptomyces Genes: Significance of the Codon Context?

Streptomycetes, Gram-positive bacteria with huge and GC-rich genomes provide an ample example of codon usage bias taken to the extreme. Particularly, in all sequenced to date streptomycete genomes leucyl codon TTA is the rarest one. It is present (usually once or twice) in 70–200 out of 7000–8000 coding sequences that make up a typical streptomycete genome. tRNA^Leu_UAA of streptomycetes, encoded by the bldA gene, has been shown to be present in mature form only after the onset of morphological differentiation and activation of secondary metabolism. Consequently, during the early stages of cell growth, the translation of genes carrying the TTA codon can be interrupted due to the absence of tRNA^Leu_UAA. Several reports show that mutations of TTA to synonymous codons in certain genes indeed relieve their expression from bldA dependence. However, the deletion of bldA does not always arrest the expression of TTA-containing genes. The nucleotides T/C downstream of TTA were suggested, in 2002, to favor TTA mistranslation. We tested this hypothesis using sizable datasets derived from individual Streptomyces genome and a subset of TTA+ genes for secondary metabolism known for their active expression. Our results revealed nucleotide biases downstream of NNA codons family, such as the preference for C and the avoidance of A. Yet, none of the observed biases was sufficient to claim a special case for TTA codon. Hence, the issue of codon context and TTA codon mistranslation in Streptomyces deserves further elaboration.Electronic supplementary materialThe online version of this article (10.1007/s12088-020-00902-6) contains supplementary material, which is available to authorized users.     

Initiator tRNA lacking 1-methyladenosine is targeted by the rapid tRNA decay pathway in evolutionarily distant yeast species

All tRNAs have numerous modifications, lack of which often results in growth defects in the budding yeast Saccharomyces cerevisiae and neurological or other disorders in humans. In S. cerevisiae, lack of tRNA body modifications can lead to impaired tRNA stability and decay of a subset of the hypomodified tRNAs. Mutants lacking 7-methylguanosine at G₄₆ (m⁷G₄₆), N₂,N₂-dimethylguanosine (m^2,2G₂₆), or 4-acetylcytidine (ac⁴C₁₂), in combination with other body modification mutants, target certain mature hypomodified tRNAs to the rapid tRNA decay (RTD) pathway, catalyzed by 5’-3’ exonucleases Xrn1 and Rat1, and regulated by Met22. The RTD pathway is conserved in the phylogenetically distant fission yeast Schizosaccharomyces pombe for mutants lacking m⁷G₄₆. In contrast, S. cerevisiae trm6/gcd10 mutants with reduced 1-methyladenosine (m¹A₅₈) specifically target pre-tRNA_i^Met(CAU) to the nuclear surveillance pathway for 3’-5’ exonucleolytic decay by the TRAMP complex and nuclear exosome. We show here that the RTD pathway has an unexpected major role in the biology of m¹A₅₈ and tRNA_i^Met(CAU) in both S. pombe and S. cerevisiae. We find that S. pombe trm6Δ mutants lacking m¹A₅₈ are temperature sensitive due to decay of tRNA_i^Met(CAU) by the RTD pathway. Thus, trm6Δ mutants had reduced levels of tRNA_i^Met(CAU) and not of eight other tested tRNAs, overexpression of tRNA_i^Met(CAU) restored growth, and spontaneous suppressors that restored tRNA_i^Met(CAU) levels had mutations in dhp1/RAT1 or tol1/MET22. In addition, deletion of cid14/TRF4 in the nuclear surveillance pathway did not restore growth. Furthermore, re-examination of S. cerevisiae trm6 mutants revealed a major role of the RTD pathway in maintaining tRNA_i^Met(CAU) levels, in addition to the known role of the nuclear surveillance pathway. These findings provide evidence for the importance of m¹A₅₈ in the biology of tRNA_i^Met(CAU) throughout eukaryotes, and fuel speculation that the RTD pathway has a major role in quality control of body modification mutants throughout fungi and other eukaryotes.     

Pleiotropic effects of cAMP on germination, antibiotic biosynthesis and morphological development in Streptomyces coelicolor

In wild-type Streptomyces coelicolor MT1110 cultures, cyclic adenosine 3′,5′ monophosphate (cAMP) was synthesized throughout the developmental programme with peaks of accumulation both during germination and later when aerial mycelium and actinorhodin were being produced. Construction and characterization of an adenylate cyclase disruption mutant (BZ1) demonstrated that cAMP facilitated these developmental processes. Although pulse-labelling experiments showed that a similar germination process was initiated in BZ1 and MT1110, germ-tube emergence was severely delayed in BZ1 and never occurred in more than 85% of the spores. Studies of growth and development on solid glucose minimal medium (SMMS, buffered or unbuffered) showed that MT1110 and BZ1 produced acid during the first rapid growth phase, which generated substrate mycelium. Thereafter, on unbuffered SMMS, only MT1110 resumed growth and produced aerial mycelium by switching to an alternative metabolism that neutralized its medium, probably by reincorporating and metabolizing extracellular acids. BZ1 was not able to neutralize its medium or produce aerial mycelium on unbuffered SMMS; these defects were suppressed by high concentrations (>1 mM) of cAMP during early growth or on buffered medium. Other developmental mutants (bldA, bldB, bldC, bldD, bldG) also irreversibly acidified this medium. However, these bald mutants were not suppressed by exogenous cAMP or neutralizing buffer. BZ1 also differentiated when it was cultured in close proximity to MT1110, a property observed in cross-feeding experiments between bald mutants and commonly thought to reflect diffusion of a discrete positively acting signalling molecule. In this case, MT1110 generated a more neutral pH environment that allowed BZ1 to reinitiate growth and form aerial mycelium. The fact that actinorhodin synthesis could be induced by concentrations of cAMP (< 20 μM) found in the medium of MT1110 cultures, suggested that it may serve as a diffusible signalling molecule to co-ordinate antibiotic biosynthesis.     

An exploratory scandium-45 NMR study into the complexation of alanine and oligopeptides

The 87.5 MHz ⁴⁵Sc NMR spectrum of 0.025 M aqueous Sc(NO₃)₃ exhibits two resonance signals, separated by ca. 25 ppm, attributable to [Sc(H₂O)₆]³⁺ and [Sc(H₂O)₅OH]²⁺. Acidification leads to a single, comparatively sharp line (W_1/2 = 160 Hz) for the hexaqua complex, the temperature dependence (temperature gradient = 0.076 ppm/deg) of which indicates that relaxation is dominated by the quadrupole mechanism. Addition of α-alanine gives rise to an additional broad signal at ca. +70 ppm (relative to [Sc(H₂O)₆]³⁺), which is assigned to a carboxylato complex [Sc(H₂O)_6−n(ala)_n]³⁺ or [Sc(H₂O)_5−nOH- (ala)_n]²⁺ (1 < n < 2). At ambient temperatures, these species are in slow exchange with the hexaqua and pentaqua-hydroxo complex, progressing through medium towards fast exchange as the temperature increases, and giving rise to an exchange contribution to relaxation. W_1/2 becomes a measure for the stability of the complexes, which increases in the order ala < (ala)₄ ∼ (ala)₂ < ala-val-leu. The pronounced stability of the latter is due to the formation of a chelate-five ring structure (participation of the NH- function of the peptide bond in coordination to Sc³⁺). 1 M aqueous ScCl₃ probably contains the two species [Sc(H₂O)₆]³⁺ and [Sc(H₂O)₅Cl]²⁺, separated by 33 ppm.     

Genetic analysis of nitrogen fixation in a tropical fast-growing Rhizobium

The Rhizobium strain ORS571, which is associated with the tropical legume Sesbania rostrata, has the property of growing in the free-living state at the expense of ammonia or N₂ as sole nitrogen source. Five mutants, isolated as unable to form colonies on plates under conditions of nitrogen fixation, were studied. All of them, which appear as Fix^- in planta, are nif mutants. With mutant 5740, nitrogenase activity of the crude extract was restored by addition of pure Mo-Fe protein of Klebsiella pneumoniae. A 13-kb BamHI DNA fragment from the wild-type strain, which hybridized with a probe carrying the nifHDK genes of K. pneumoniae, was cloned in vector pRK290 to yield plasmid pRS1. The extent of homology between the probe and the BamHI fragment was estimated at 4 kb and hybridization with K. pneumoniae nifH, nifK, and possibly nifD was detected. The pRS1 plasmid was introduced into the sesbania rhizobium nif mutants. Genetic complementation was observed with strain 5740(pRS1) both in the free-living state and in planta. It thus appears that biochemistry and genetics of nitrogen fixation in this particular Rhizobium strain can be performed with bacteria grown under non-symbiotic conditions.     

Glucose repression in Streptomyces coelicolor A3(2): a likely regulatory role for glucose kinase

The glucose kinase gene (glkA-ORF3) of Streptomyces coelicolor A3(2) plays an essential role in glucose utilisation and in glucose repression of a variety of genes involved in the utilisation of alternative carbon sources. These genes include dagA, which encodes an extracellular agarase that permits agar utilisation. Suppressor mutants of glkA-ORF3 deletion strains capable of utilising glucose (Glc⁺) arise at a frequency of about 10^?5 on prolonged incubation. The Glc⁺ phenotype of the mutants is reversible (at a frequency of about 10^?3) and reflects either the activation of a normally silent glucose kinase gene or the modification of an existing sugar kinase. Although the level of glucose kinase activity in the Glc⁺ supressor mutants is similar to that in the glkA ⁺ parental strain, glucose repression of dagA remains defective. Expression of the glucose kinase gene of Zymomonas mobilis in glkA-ORF3 mutants restored glucose utilisation, but not glucose repression of dagA. Over-expression of glkA-ORF3 on a high-copy-number plasmid failed to restore glucose repression of dagA in glkA-ORF3 mutants and led to loss of glucose repression of dagA in a glkA ⁺ strain. These results suggest that glucose phosphorylation itself is not sufficient for glucose repression and that glkA-ORF3 plays a specific regulatory role in triggering glucose repression in S. coelicolor A3(2).     

Phage-mediated cloning of bldA, a region involved in Streptomyces coelicolor morphological development, and its analysis by genetic complementation.

Streptomyces coelicolor bald (bld) mutants form colonies of vegetative substrate mycelium, but do not develop aerial hyphae or spore chains. The bldA strains form none of the four antibiotics known to be produced by the parent strain. With a vector derived from the temperate bacteriophage phi C31, a 5.6-kilobase fragment of wildtype DNA was cloned which restored sporulation to five independent bldA mutants when lysogenized with the recombinant phage. The cloned gene(s) was dominant over the mutant alleles. Phage integration by recombination of the cloned bldA+ DNA with the bldA region of each mutant produced mainly sporulating colonies, presumably heterozygous bldA+/bldA partial diploids for the insert DNA. However, a minority of these primary transductants were bald and were apparently homozygous bldA/bldA mutant partial diploids, formed by some homogenetization process. The phages released from the bald lysogens carried bldA mutations and were used to show that bldA+ sequences had been cloned and that fine mapping of the region could be performed.     

Hydrogen sulfide formation as well as ethanol production in different media by cysND- and/or cysIJ-inactivated mutant strains of Zymomonas mobilis ZM4

Many bacteria reduce inorganic sulfate to sulfide to satisfy their need for sulfur, one of the most important elements for biological life. But little is known about the metabolic pathways involving hydrogen sulfide (H₂S) in mesophilic bacteria. By genomic sequence analysis, a complete set of genes for the assimilatory sulfate reduction pathway has been identified in the ethanologen Zymomonas mobilis. In this study, the first ATP sulfurylase- and final sulfite reductase-encoding genes cysND and cysIJ, respectively, in the putative pathway from sulfate to sulfite in Z. mobilis ZM4 was singly or doubly inactivated by homologous recombination and a site-specific FLP-FRT recombination. The resultant mutants, ?cysND, ?cysIJ and ?cysND-cat?cysIJ, were unable to produce detectable H₂S in glucose or sucrose-containing rich medium and sweet sorghum juice, in which the wild-type ZM4 produced detectable H₂S. While adding sulfite (SO₃ ^2?) into media impaired the growth of the mutants and ZM4 to varying degrees, the sulfite restored the H₂S formation in the ?cysND in the above media, but not in the ?cysIJ and ?cysND-cat?cysIJ mutants. Although it seemed that the inactivation of cysND and cysIJ did not exert a significant negative effect on the cell growth at least in glucose or sucrose medium, the ethanol production of all mutants was inferior to that of ZM4 in sucrose medium and sweet sorghum juice. In addition, adding l-cysteine to glucose-containing rich media restored H₂S formation of all mutants, indicating the existence of another pathway for producing H₂S in Z. mobilis. All these results would help to further elucidate the metabolic pathways involving H₂S in Z. mobilis and exploit the biotechnological applications of this industrially important bacterium.     

Disruption of the Pseudomonas aeruginosa Tat system perturbs PQS-dependent quorum sensing and biofilm maturation through lack of the Rieske cytochrome bc1 sub-unit

Extracellular DNA (eDNA) is a major constituent of the extracellular matrix of Pseudomonas aeruginosa biofilms and its release is regulated via pseudomonas quinolone signal (PQS) dependent quorum sensing (QS). By screening a P. aeruginosa transposon library to identify factors required for DNA release, mutants with insertions in the twin-arginine translocation (Tat) pathway were identified as exhibiting reduced eDNA release, and defective biofilm architecture with enhanced susceptibility to tobramycin. P. aeruginosa tat mutants showed substantial reductions in pyocyanin, rhamnolipid and membrane vesicle (MV) production consistent with perturbation of PQS-dependent QS as demonstrated by changes in pqsA expression and 2-alkyl-4-quinolone (AQ) production. Provision of exogenous PQS to the tat mutants did not return pqsA, rhlA or phzA1 expression or pyocyanin production to wild type levels. However, transformation of the tat mutants with the AQ-independent pqs effector pqsE restored phzA1 expression and pyocyanin production. Since mutation or inhibition of Tat prevented PQS-driven auto-induction, we sought to identify the Tat substrate(s) responsible. A pqsA::lux fusion was introduced into each of 34 validated P. aeruginosa Tat substrate deletion mutants. Analysis of each mutant for reduced bioluminescence revealed that the primary signalling defect was associated with the Rieske iron-sulfur subunit of the cytochrome bc₁ complex. In common with the parent strain, a Rieske mutant exhibited defective PQS signalling, AQ production, rhlA expression and eDNA release that could be restored by genetic complementation. This defect was also phenocopied by deletion of cytB or cytC₁. Thus, either lack of the Rieske sub-unit or mutation of cytochrome bc₁ genes results in the perturbation of PQS-dependent autoinduction resulting in eDNA deficient biofilms, reduced antibiotic tolerance and compromised virulence factor production.     

A surface active protein involved in aerial hyphae formation in the filamentous fungus Schizophillum commune restores the capacity of a bald mutant of the filamentous bacterium Streptomyces coelicolor to erect aerial structures

The filamentous bacterium Streptomyces coelicolor undergoes a complex process of morphological differentiation involving the formation of a dense lawn of aerial hyphae that grow away from the colony surface into the air to form an aerial mycelium. Bald mutants of S. coelicolor, which are blocked in aerial mycelium formation, regain the capacity to erect aerial structures when exposed to a small hydrophobic protein called SapB, whose synthesis is temporally and spatially correlated with morphological differentiation. We now report that SapB is a surfactant that is capable of reducing the surface tension of water from 72 mJ m^?2 to 30 mJ m^?2 at a concentration of 50 μg ml^?1. We also report that SapB, like the surface-active peptide streptofactin produced by the species S. tendae, was capable of restoring the capacity of bald mutants of S. tendae to erect aerial structures. Strikingly, a member (SC3) of the hydrophobin family of fungal proteins involved in the erection of aerial hyphae in the filamentous fungus Schizophyllum commune was also capable of restoring the capacity of S. coelicolor and S. tendae bald mutants to erect aerial structures. SC3 is unrelated in structure to SapB and streptofactin but, like the streptomycetes proteins, the fungal protein is a surface active agent. Scanning electron microscopy revealed that aerial structures produced in response to both the bacterial or the fungal proteins were undifferentiated vegetative hyphae that had grown away from the colony surface but had not commenced the process of spore formation. We conclude that the production of SapB and streptofactin at the start of morphological differentiation contributes to the erection of aerial hyphae by decreasing the surface tension at the colony surface but that subsequent morphogenesis requires additional developmentally regulated events under the control of bald genes.     

Cloning and expression of a tylosin resistance gene from a tylosin-producing strain of Streptomyces fradiae

Summary A gene conferring high-level resistance to tylosin in Streptomyces lividans and Streptomyces griseofuscus was cloned from a tylosin-producing strain of Streptomyces fradiae. The tylosin-resistance (Tyl^r) gene (tlrA) was isolated on five overlapping DNA fragments which contained a common 2.6 Kb KpnI fragment. The KpnI fragment contained all of the information required for the expression of the Tyl^r phenotype in S. lividans and S. griseofuscus. Southern hybridization indicated that the sequence conferring tylosin resistance was present on the same 5 kb SalI fragment in genomic DNA from S. fradiae and several tylosin-sensitive (Tyl^s) mutants. The cloned tlrA gene failed to restore tylosin resistance in two Tyl^s mutants derived by protoplast formation and regeneration, and it restored partial resistance in a Tyl^s mutant obtained by N-methyl-N-nitro-N-nitrosoguanidine (MNNG) mutagenesis. The tlrA gene conferred resistance to tylosin, carbomycin, niddamycin, vernamycin-B and, to some degree, lincomycin in S. griseofuscus, but it had no effect on sensitivity to streptomycin or spectinomycin, suggesting that the cloned gene is an MLS (macrolide, lincosamide, streptogramin-B)-resistance gene. Twenty-eight kb of S. fradiae DNA surrounding the tlrA gene was isolated from a genomic library in bacteriophage Charon 4. Introduction of these DNA sequence into S. fradiae mutants blocked at different steps in tylosin biosynthesis failed to restore tylosin production, suggesting that the cloned Tyl^r gene is not closely linked to tylosin biosynthetic genes.     

Regulation of nitrogen fixation in Rhizobium spp. Isolation of mutants of Rhizobium trifulii which induce nitrogenase activity

This communication describes the isolation and characterization of mutants of Rhizobium trifolii which can induce nitrogenase activity in defined liquid medium. Two procedures were used for the isolation of these mutants from R. trifolii strain DT-6: (1) following chemical mutagenesis, slow growin mutants were selected which were unable to utilize NH₄⁺ as sole source of nitrogen; (2) as spontaneous mutants resistant to the glutamate analogue L-methionine-DL-sulfoximine.Mutants (DT-71, DT-125) isolated by these procedures induced nitrogenase activity in the free-living state, whereas the parent strain lacked this property. Induction of nitrogenase activity in these mutants occurred during the late exponential phase of growth when the rate of protein synthesis was decreasing. The addition of NH₄⁺ to a medium containing glutamate as the nitrogen-source resulted in a 50–70% reduction (repression?) of nitrogenase activity; in contrast, the rate of protein synthesis or the rate of respiration was not influenced by exogenous NH₄⁺.Biochemistry analysis showed that these mutants (strains DT-71 and DT-125) have defects in both nitrogen and carbon metabolism. The levels of glutamate synthase (both NADP⁺-and NAD⁺-dependent activities) and glutamate dehydrogenase (NAD⁺-dependent activity) were markedly lower. In addition, the mutants were found to have no detectable ribitol dehydrogenase or β-galactosidase activity. These findings are discussed in relation to a mechanism of regulation of symbiotic nitrogen fixation.     

Molecular and functional analysis of the ribosomal L11 and S12 protein genes (rplK and rpsL) of Streptomyces coelicolor A3(2)

A RelC deletion mutant, KO-100, of Streptomyces coelicolor A3(2) has been isolated from a collection of spontaneous thiostrepton-resistant mutants. KO-100 grows as vigorously as the parent strain and possesses a 6-bp deletion within the rplK, previously termed relC. When the wild-type rplK gene was propagated on a low-copy-number vector in mutant KO-100, the ability to produce ppGpp, actinorhodin and undecylprodigiosin, which had been lost in the RelC mutant, was completely restored. Allele replacement by gene homogenotization demonstrated that the RelC mutation is responsible for the resistance to thiostrepton and the inactivation of ppGpp, actinorhodin and undecylprodigiosin production. Western blotting showed that ribosomes from the RelC mutant KO-100 contain only one-eighth the amount of L11 protein found in ribosomes of the parent strain. The impairment of antibiotic production in KO-100 could be rescued by the introduction of mutations that confer resistance to streptomycin (str), which result in alteration of Lys-88 in ribosomal protein S12 to Glu or Arg. No accompanying restoration of ppGpp synthesis was detected in these RelC str double mutants.     

Glucosylation of Lipopolysaccharide in Salmonella: Mutants Negative for O Antigen Factor 122

In salmonella O group B, the O antigen factor 12₂ is created by glucosylation at C4 of the galactose units of the O side chains; phage-determined glucosylation at C6 of these galactose units yields factor 1. 12₂-negative mutants were isolated from 12₂⁺ (stable) parents (genetically oafR^+st). All 20 mutants studied were stable in respect of their 12₂ character. In eight of them, lysogenization by phage P22 resulted in the appearance of the 12₂ factor—these were interpreted to be defective in the enzyme(s) needed to synthesize the glucose-lipid intermediate that participates in both 12₂- and 1-specific glucosylation; the corresponding cistron was termed oafE. This “complementation” assay also demonstrated that the P22 genome can determine the synthesis of this glucose-lipid intermediate. The 12₂ character in the oafE⁻ mutants became variable after P22 lysogenization, corresponding to factor 1 variation normally determined by this phage. In the remaining 12 mutants, P22 caused the appearance of a variable factor 1, but not of 12₂. The lesion in all the 12₂ mutants was mapped close to the gene purE at min 19 of the Salmonella map. This is the same area where the locus oafR that controls the variation (stable or variable, + or −) of 12₂ had been previously mapped. The results suggest the presence of a 12₂ glucosylation operon in this region.