Insertional inactivation of genes encoding eukaryotic type serine/threonine protein kinases in cyanobacterium Synechocystis sp. PCC 6803

Synechocystis sp. PCC 6803 mutants, in which one of the eukaryotic-type serine/threonine protein kinase genes pknD, pknE, pknG, and pknH was inactivated, were obtained by insertion mutagenesis. None of these mutants differed phenotypically from the wild-type strain, indicating that the pknD, pknE, pknG, and pknH genes are not of crucial importance for the photoautotrophically grown cyanobacterium. Mutant with the inactivated pknE gene was resistant to L-methionine-D,L-sulfoximine and especially to methylamine. The resistance was neither due to the impaired transport of these compounds nor to the inhibition of the production of toxic gamma-glutamylmethylamide from methylamine. The data presented suggest that resistance to methylamine may be associated with alterations in the regulation of the glutamine synthetase system and that the PknE protein kinase may be involved in the regulation of nitrogen metabolism in the cyanobacterium studied.     

PknG supports mycobacterial adaptation in acidic environment

Mycobacterium tuberculosis (Mtb), causative agent of human tuberculosis (TB), has the remarkable ability to adapt to the hostile environment inside host cells. Eleven eukaryotic like serine-threonine protein kinases (STPKs) are present in Mtb. Protein kinase G (PknG) has been shown to promote mycobacterial survival inside host cells. A homolog of PknG is also present in Mycobacterium smegmatis (MS), a fast grower, non-pathogenic mycobacterium. In the present study, we have analyzed the role of PknG in mycobacteria during exposure to acidic environment. Expression of pknG in MS was decreased in acidic medium. Recombinant MS ectopically expressing pknG (MS-G) showed higher growth in acidic medium compared to wild type counterpart. MS-G also showed higher resistance upon exposure to 3.0 pH and better adaptability to acidic pH. Western blot analysis showed differential threonine but not serine phosphorylation of cellular proteins in MS at acidic pH which was restored by ectopic expression of pknG in MS. In Mtb H37Ra (Mtb-Ra), expression of pknG was increased at acidic pH. We also observed decreased expression of pknG in MS during infection in macrophages while the expression of pknG in Mtb-Ra was increased in similar conditions. Taken together, our data strongly suggests that pknG regulates growth of mycobacteria in acidic environment and is differentially transcribed in MS and Mtb under these conditions.     

Overexpression of pknE blocks heterocyst development in Anabaena sp. strain PCC 7120

The upstream intergenic regions for each of four genes encoding Ser/Thr kinases, all2334, pknE (alr3732), all4668, and all4838, were fused to a gfpmut2 reporter gene to determine their expression during heterocyst development in the cyanobacterium Anabaena (Nostoc) sp. strain PCC 7120. P(pknE)-gfp was upregulated after nitrogen step-down and showed strong expression in differentiating cells. Developmental regulation of pknE required a 118-bp upstream region and was abolished in a hetR mutant. A pknE mutant strain had shorter filaments with slightly higher heterocyst frequency than did the wild type. Overexpression of pknE from its native promoter inhibited heterocyst development in the wild type and in four mutant backgrounds that overproduce heterocysts. Overexpression of pknE from the copper-inducible petE promoter did not completely inhibit heterocyst development but caused a 24-h delay in heterocyst differentiation and cell bleaching 4 to 5 days after nitrogen step-down. Strains overexpressing pknE and containing P(hetR)-gfp or P(patS)-gfp reporters failed to show developmental regulation of the reporters and had undetectable levels of HetR protein. Genetic epistasis experiments suggest that overexpression of pknE blocks HetR activity or downstream regulation.     

Biochemical and genetic studies on two different types of erythromycin resistant mutants of Escherichia coli with altered ribosomal proteins

Summary Ribosomes from nine E. coli mutants with high level resistance to the antibiotic erythromycin were isolated and their proteins were compared with those of the parental strains by two-dimensional polyacrylamide gel electrophoresis, by carboxymethylcellulose column chromatography and by immunological techniques. Two 50S proteins were found to be altered in the mutants: either L 4 or L 22.Ribosomes with an altered L4 protein bound erythromycin rather poorly and the formation of N-acetylphenylalanyl puromycin was drastically decreased. On the other handribosomes with an altered L22 protein bound erythromycin as efficiently as wild type ribosomes and their puromycin reaction was at least as good as that of wild type ribosomes.Transduction experiments showed that the mutations affecting both proteins, L4 and L22, are located very close to the str and spc genes, nearer to the spc than to str gene.Paper No. 61 on Ribosomal Proteins. Preceding paper is by Hasenbank et al., Molec. gen. Genet., 127, 1–18 (1973).Communicated by E. Bautz     

The <Emphasis Type="Italic">Arabidopsis</Emphasis><Emphasis Type="Italic">KLUNKER</Emphasis> gene controls cell shape changes and encodes the AtSRA1 homolog

The analysis of a group of seven trichome mutants in Arabidopsis, which all show distorted trichomes along with severe actin defects has revealed insight into the role of the actin cytoskeleton in cell shape control. Four of the corresponding genes encode components of a protein complex, the ARP2/3 complex that stimulates the production of fine actin at active growth sites. In this study, we show that another member of the distorted group, KLUNKER (KLK), encodes the AtSRA1 homolog of Arabidopsis and that klk mutants show a similar range of cell shape defects to those of arp2/3 mutants. In animals, SRA1 regulates the activity of the ARP2/3-regulating WAVE-HSPC300 complex in a Rho-dependent manner. Our findings provide evidence that a Rho/ARP2/3 regulation pathway exists in plants.     

Genetic evidence for the functional redundancy of the calcineurin-and Mpk1-mediated pathways in the regulation of cellular events important for growth inSaccharomyces cerevisiae

Saccharomyces cerevisiae mutants which exhibit phenotypes (calcium resistance and vanadate sensitivity) similar to those of calcineurin-deficient mutants were isolated. The mutants were classified into four complementation groups (crv1,2,3 and4).crv1 was allelic tocnb1, a mutation in the regulatory subunit of calcineurin. The nucleotide sequences ofCRV2 andCRV3 genes which complemented thecrv2 andcrv3 mutations, respectively, are identical to those ofBCK1/SLK1/SKC1/SSP31 andMPK1/SLT2, respectively, which are both involved in the MAP kinase cascade. A calcineurin-deletion mutation (cnb1), which by itself has no detectable effect on growth and morphology, enhanced some phenotypes (slow growth and morphological abnormality) ofcrv2 andcrv3 mutants. These phenotypes ofcrv2 andcrv3 mutants were partially suppressed by Ca²⁺ or by overproduction of the calcineurin subunits (Cmp2 and Cnb1). Like the calcineurin-deficient mutant,crv2 andcrv3 mutants were defective in recovery from -factor-induced growth arrest. The defect in recovery of the cnb1 mutant was suppressed by overexpression ofMPK1. These results indicated that the calcineurin-mediated and the Mpk1- (Bck1-) mediated signaling pathways act in parallel to regulate functionally redundant cellular events important for growth.     

Molecular analysis of psb A mutations responsible for various herbicide resistance phenotypes in Synechocystis 6714

Mutations conferring herbicide resistance in 3 mutant strains of the cyanobacterium Synechocystis 6714 have been characterized by gene cloning and sequencing. The mutants display very different phenotypes: DCMU-II_A is DCMU-resistant and atrazine-resistant, DCMU-II_B is DCMU-resistant and atrazine-sensitive, and Az-V is DCMU-sensitive, atrazine-resistant and presents particular photoinhibition properties. These mutants were originally obtained either by one-step selection (DCMU-II_A) or by two-step selection (DCMU-II_B and Az-V). psbA copies carrying herbicide resistance have been identified by transformation experiments as psbAI in all cases. Sequences of the psbAI copy of each mutant have been compared to the wild-type sequence. In the single mutant DCMU-II_A, a point mutation at codon 264 (SerAla) results in resistance to both DCMU and atrazine. In the double mutants DCMU-II_B and Az-V, two point mutations were found. DCMU-II_B was derived from DCMU-II_A and had acquired a second mutation at codon 255 (PheLeu) resulting in a slight increase in DCMU resistance and complete abolition of atrazine resistance. Az-V contains two changes at codons 211 (PheSer) and 251 (AlaVal) resulting in high atrazine resistance but only slight DCMU resistance.Abbreviations DCMU: 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PSII: photosystem II     

SLK1, a yeast homolog of MAP kinase activators, has a RAS/cAMP-independent role in nutrient sensing

The Saccharomyces cerevisiae SLK1 protein is implicated in nutrient sensing and growth control. Under nutrient-limiting conditions, slk1 mutants fail to undergo cell cycle arrest. The role of the SLK1 protein in nutrient sensing was examined with respect to the cAMP-dependent protein kinase (PKA) pathway, which has a well characterized role in growth control in yeast, and by the analysis of dominant SLK1 alleles that affect the nutrient response of wild-type cells. Interactions with the PKA pathway were examined by phenotypic analysis of double mutants of slk1 and various PKA pathway mutants. Combining the slk1- mutation with a mutation that is thought constitutively activate the PKA pathway, pde2, resulted in enhanced growth control defects. The combination of slk1- with mutations that inhibit the PKA pathway, cdc25 and ras1 ras2, failed to alleviate the slk1 cell cycle arrest defect and lowered the permissive temperature for growth. Furthermore bcy1 tpk1 tpk2 tpk3 ^w (bcyl tpk ^w) mutants, which have constitutive, low-level, cAMP-independent kinase activity, exhibit nutrient sensing, which is eliminated in the slk1 bcy1 tpk ^w mutants. These results implicated SLK1 in PKA-independent growth control in yeast. The amino-terminal, noncatalytic region of the SLK1 protein may be important in the regulation of SLK1 function in growth control. Overexpression of this region caused starvation sensitivity in wild-type cells by interfering with SLK1 protein function.     

Mutants ofMethylobacterium organophilum unable to synthesize PQQ

The phenotype of mutants unable to synthesize PQQ is analyzed for different categories of methylotrophic bacteria. The advantages offered by strains dissimilating methylamine through methylated amino-acids are discussed. InM.organophilum, 40% of the mutants unable to grow in methanol medium but with normal methylamine utilization, were affected in PQQ metabolism. The genetic properties ofM.organophilum useful to study PQQ mutants are discussed, mainly the use of pSUP106 to create insertion mutations in the bacterial chromosome and to replace wild-type genes by modified genes. An example is given of the possibility to create R plasmids containing large fragments ofM.organophilum DNA. Some physiological properties of a PQQ mutant are described, regarding growth kinetics, PQQ uptake and accumulation.     

Round-cell mutants of Salmonella typhimurium produced by transposition mutagenesis: Lethality of rodA and mre mutations

Summary Thirty-three insertions of transposon Tn10l6l7 into genes involved in the control of rod cell shape were isolated in Salmonella typhimurium by the characteristic glossy appearance of colonies composed of spherical cells. Genetic tests demonstrated that 25 (76%) were insertions in the rodA gene, 7 (21 %) were mre mutants, and 1 (3%) was a divD mutant. No insertion in the pbpA gene were found. Insertions in cell shape genes only appeared when strains displaying resistance to mecillinam (not caused by -lactamase production) were employed. Neither rodA nor mre insertions could be transduced to wild-type strains but they were normally accepted by mecillinam-resistant derivatives and by cya and crp mutants, which, unlike the corresponding Escherichia coli strains, did not display resistance to mecillinam. On the other hand, the divD insertion could be efficiently transduced to any strain. It is concluded that the rodA, mre, and divD genes are involved in the control of rod cell shape but, in addition, the RodA and Mre products perform some function(s) that is essential for wild-type cells but dispensable for some mecillinam-resistant strains, and for cya and crp mutants.     

MauE and MauD proteins are essential in methylamine metabolism of Paracoccus denitrificans

Synthesis of enzymes involved in methylamine oxidation via methylamine dehydrogenase (MADH) is encoded by genes present in the mau cluster. Here we describe the sequence of the mauE and mauD genes from Paracoccus denitrificans as well as some properties of mauE and mauD mutants of this organism. The amino acid sequences derived from the mauE and mauD genes showed high similarity with their counterparts in related methylotrophs. Secondary structure analyses of the amino acid sequences predicted that MauE is a membrane protein with five transmembrane-spanning helices and that MauD is a soluble protein with an N-terminal hydrophobic tail. Sequence comparison of MauD proteins from different organisms showed that these proteins have a conserved motif, Cys-Pro-Xaa-Cys, which is similar to a conserved motif found in periplasmic proteins that are involved in the biosynthesis of bacterial periplasmic enzymes containing haem c and/or disulphide bonds. The mauE and mauD mutant strains were unable to grow on methylamine but they grew well on other C₁-compounds. These mutants grown under MADH-inducing conditions contained normal levels of the natural electron acceptor amicyanin, but undetectable levels of the -subunit and low levels of the -subunit of MADH. It is proposed, therefore, that MauE and MauD are specifically involved in the processing, transport, and/or maturation of the -subunit and that the absence of each of these proteins leads to production of a non-functional -subunit which becomes rapidly degraded.     

ThehupB gene of theAzotobacter chroococcum hydrogenase gene cluster is involved in nickel metabolism

InAzotobacter chroococcum the hydrogenase gene (hup) cluster spans about 14 kb of DNA. The genes coding for the small and large subunits,hupSL, are located at the 5 end, and a cluster of genes,hupABYCDE, resembling theEscherichia coli hyp operon, is located at the 3 end. In this study, we determined the effect of adding nickel to the medium used for the growth ofhup mutants. Hydrogenase activity was restored tohupA andhupB mutants, but nothupY, hupD, orhupE mutants, by the addition of nickel to the growth medium, suggesting that the products ofhupA andhupB are somehow involved in nickel metabolism. The restoration of hydrogenase activity to thehupB mutant required protein synthesis.     

C1 metabolism inParacoccus denitrificans: Genetics ofParacoccus denitrificans

Paracoccus denitrificans is able to grow on the C₁ compounds methanol and methylamine. These compounds are oxidized to formaldehyde which is subsequently oxidized via formate to carbon dioxide. Biomass is produced by carbon dioxide fixation via the ribulose biphosphate pathway. The first oxidation reaction is catalyzed by the enzymes methanol dehydrogenase and methylamine dehydrogenase, respectively. Both enzymes contain two different subunits in an ₂₂ configuration. The genes encoding the subunits of methanol dehydrogenase (moxF andmoxI) have been isolated and sequenced. They are located in one operon together with two other genes (moxJ andmoxG) in the gene ordermoxFJGI. The function of themoxJ gene product is not yet known.MoxG codes for a cytochromec _551i , which functions as the electron acceptor of methanol dehydrogenase. Both methanol dehydrogenase and methylamine dehydrogenase contain PQQ as a cofactor. These so-called quinoproteins are able to catalyze redox reactions by one-electron steps. The reaction mechanism of this oxidation will be described. Electrons from the oxidation reaction are donated to the electron transport chain at the level of cytochromec. P. denitrificans is able to synthesize at least 10 differentc-type cytochromes. Five could be detected in the periplasm and five have been found in the cytoplasmic membrane. The membrane-bound cytochromec ₁ and cytochromec ₅₅₂ and the periplasmic-located cytochromec ₅₅₀ are present under all tested growth conditions. The cytochromesc _551i andc _553i , present in the periplasm, are only induced in cells grown on methanol, methylamine, or choline. The otherc-type cytochromes are mainly detected either under oxygen limited conditions or under anaerobic conditions with nitrate as electron acceptor or under both conditions. An overview including the induction pattern of allP. denitrificans c-type cytochromes will be given. The genes encoding cytochromec ₁, cytochromec ₅₅₀, cytochromec _551i , and cytochromec _553i have been isolated and sequenced. By using site-directed mutagenesis these genes were mutated in the genome. The mutants thus obtained were used to study electron transport during growth on C₁ compounds. This electron transport has also been studied by determining electron transfer rates inin vitro experiments. The exact pathways, however, are not yet fully understood. Electrons from methanol dehydrogenase are donated to cytochromec _551i . Further electron transport is either via cytochromec ₅₅₀ or cytochromec _553i to cytochromeaa ₃. However, direct electron transport from cytochromec _551i to the terminal oxidase might be possible as well. Electrons from methylamine dehydrogenase are donated to amicyanin and then via cytochromec ₅₅₀ to cytochromeaa ₃, but other routes are used also.P. denitrificans is studied by several groups by using a genetic approach. Several genes have already been cloned and sequenced and a lot of mutants have been isolated. The development of a host/vector system and several techniques for mutation induction that are used inP. denitrificans genetics will be described.     

Induced transposition of Ds by a stable Ac in crosses of transgenic tobacco plants

Summary Rhizoxin and ansamitocin P-3 (a maytansinoid compound), potent inhibitors of mammalian brain tubulin assembly, inhibit growth of a variety of fungi including Aspergillus nidulans. Mutants of A. nidulans, benA10 which is a benomyl resistant -tubulin gene mutant and tubAl which is a benomyl supersensitive a-tubulin gene mutant, were both sensitive to rhizoxin and ansamitocin P-3 to the same extent as wild-type strains. We isolated 18 rhizoxin resistant mutants of A. nidulans. All of these mutants were cross-resistant to ansamitocin P-3, but not to benzimidazole antimitotic drugs. These mutants mapped to two loci, rhiA and rhiB, and all of those with high resistance mapped to rhiA. The fact that the protein extracts of rhiA mutants lost rhizoxin binding affinity and that rhiA was closely linked to benA, the major -tubulin gene in A. nidulans, indicated that rhiA must be a structural gene for -tubulin and that rhiA mutants are a new class of -tubulin gene mutants. All of this suggested that, in A. nidulans, these antimitotic drugs bind to -tubulin, and that rhizoxin and ansamitocin P-3 share the same binding site but the site does not overlap with the benzimidazole binding site. Protein extracts from a rhiB mutant retained rhizoxin binding affinity, therefore this rhizoxin resistance mechanism should not be a tubulin mediated process.     

Mutants of Cyanobacterium Synechocystis sp. PCC 6803 with Insertional Inactivation of the sodB Gene Encoding Fe-Superoxide Dismutase

The sodB gene encoding the only superoxide dismutase (Fe-SOD) in cells of the cyanobacterium Synechocystis sp. PCC 6803 was inactivated with gentamycin resistance aacC1 marker insertions located in the direct or reverse direction in the sodB gene. The corresponding sodB12 and sodB22 mutants are characterized by the complete absence of superoxide dismutase activity and the loss of viability upon standard photoautotrophic cultivation. Mutant cells can grow under conditions of a decreased illumination intensity and upon addition of NaHCO₃ with catalase or bovine serum albumin in the growth medium. The sodB22 mutant is auxotrophic for leucine due to the polar effect of insertion into the sodB gene on the downstream leuB gene controlling leucine biosynthesis. These data suggest that Fe-SOD is very important for providing resistance of Synechocystis 6803 cells to oxidative stress and thatsodB and leuB genes are organized into a single operon.     

The nature of ompA mutants of Escherichia coli K12 exhibiting temperature-sensitive bacteriophage resistance

Summary A class of ompA mutants of Escherichia coli, exhibiting temperature-sensitive resistance towards phages using the OmpA protein as receptor, was analysed. The mutants produce detectable levels of the protein at 42°C but not at 30°C (Manning and Reeves 1976). They were found to have a deletion (one isolate) or insertions (three isolates) upstream of the coding part of the ompA gene. Several previously characterized mutants possessing insertions or a deletion in the non-translated 5 area of the gene also exhibited a similar temperature-sensitive phage resistance. This cold-sensitive phenotype is explained in terms of the recent discovery that the stability of ompA mRNA is regulated by the rate of cell growth (Nilsson et al. 1984).     

Efficient transformation of Claviceps purpurea using pyrimidine auxotrophic mutants: cloning of the OMP decarboxylase gene

Summary A homologous transformation system was developed for the phytopathogenic fungus Claviceps purpurea. Orotidine-5-monophosphate decarboxylase (OMPD)-deficient mutants were obtained by UV mutagenesis and selection for resistance against 5-fluoroorotate. These mutants could be complemented well by the corresponding genes of Aspergillus niger (pyrA) and Neurospora crassa (pyr4), yielding significantly higher transformation rates (and lower copy numbers per transformant) than the phleomycin resistance system. The homologous OMPD gene was isolated from a lambda genomic library by heterologous hybridization with the pyr4 gene of N. crassa, identified by complementation of Aspergillus and Claviceps mutants, and used to confirm homologous integration in Claviceps. The pyr transformation system also proved to be very efficient in cotransformation experiments using the bacterial -glucuronidase gene (uidA) as a reporter gene, which was also efficiently expressed during the parasitic cycle: honeydew produced by plants infected with pyr/uidA cotransformants was shown to contain significant levels of -glucuronidase activity.     

Permeability of ammonia,methylamine and ethylamine in the cyanobacterium,Synechococcus R-2 (Anacystis nidulans) PCC 7942

Summary Permeabilities of ammonia (NH₃), methylamine (CH₃NH₂) and ethylamine (CH₃CH₂NH₂) in the cyanobacterium (cyanophyte)Synechococcus R-2 (Anacystis nidulans) have been measured. Based on net uptake rates of DCMU (dichlorophenyldimethylurea) treated cells, the permeability of ammonia was 6.44±1.22 m sec^–1 (n=13). The permeabilities of methylamine and ethylamine, based on steady-state¹⁴C labeling were more than ten times that of ammonia (P _methylamine=84.6±9.47 m sec^–1 (76),P _ethylamine=109±11 m sec^–1 (55)). The apparent permeabilities based on net uptake rates of methylamine and ethylamine uptake were significantly lower, but this effect was partially reversible by ammonia, suggesting that net amine fluxes are rate limited by proton fluxes to an upper limit of about 700 nmol m^–2 sec^–1. Increasing concentrations of amines in alkaline conditions partially dissipated the pH gradient across the cell membrane, and this property could be used to calculate the relative permeabilities of different amines. The ratio of ethylamine to methylamine permeabilities was not significantly different from that calculated from the direct measurements of permeabilities; ammonia was much less effective in dissipating the pH gradient across the cell membrane than methylamine or ethylamine. An apparent permeability of ammonia of 5.7±0.9 m sec^–1 could be calculated from the permeability ratio of ammonia to methylamine and the experimentally measured permeability of methylamine. The permeability properties of ammonia and methylamine are very different; this poses problems in the interpretation of experiments where¹⁴C-methylamine is used as an ammonia analogue.     

Cooperative function of the CHD5-like protein Mdm39p with a P-type ATPase Spf1p in the maintenance of ER homeostasis in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Spf1p is a P-type ATPase that is mainly localized to the endoplasmic reticulum (ER) in Saccharomyces cerevisiae. The protein is involved in the maintenance of ion homeostasis in the ER. To investigate the intracellular role of Spf1p in more detail, we performed a genetic screen for mutations that lead to synthetic lethality in combination with a disruption of SPF1; the mutations identified have been termed lws (for lethal with spf1) mutations. Mutant alleles of five LWS genes (MDM39, RIC1, LAS21, TUP1 and BTS1) were recovered. The identification of these genes provides clues to the physiological relationships between Spf1p function and the secretory pathway. Among the five genes identified, MDM39 encodes a membrane protein that is similar to the protein CHD5/WRB, which is involved in the pathogenesis of Down syndrome-associated congenital heart disease in humans. We localized Mdm39p to the ER. The mdm39 mutant exhibited defects in glycosylation, cell wall organization and the unfolded protein response. It also showed calcium-related phenotypes and synthetic lethal interactions with deletion mutations in other LWS genes. Our findings imply a homeostatic role for Mdm39p, which may be related to the regulation of calcium ion fluxes in the ER, and is indispensable for mutants that lack Spf1p.     

Non-random distribution of transduction termini in transductants from the integrated R plasmid, R100-1

Summary Tra ⁺and tra ^–derivatives of drug resistance plasmid, R100-1, were isolated by phage P1 from an Hfr donor with integrated R100-1 and then analyzed by complementation tests with tra ^–point mutants of Flac. Tra ⁺derivatives of R100-1 carrying tetracycline resistance alone and those carrying all six drug-resistance genes could support transfer of tra ^–point mutants of Flac except Flac traJ, whereas all of tra ^–derivatives of R100-1 failed to complement any one of tra ^–point mutants of Flac. This suggests that these tra ^–derivatives of R100-1 carrying tetracycline resistance gene are deleted for all the transfer genes impaired in the Flac point mutants tested. We assume a hot point, probably a specific base sequence similar to an IS element, at the left of the tetracycline gene (Fig. 1) becomes a transduction terminus in transduction of the integrated R100-1 by phage P1. Complementation analysis of tra ^–derivatives carrying five resistance genes except the tetracycline gene led us to a supposition that a gene(s), probably analogous to traJ of the F plasmid, is located on R100-1 near the tetracycline gene which plays an important regulatory role for self-transfer as well as for the complementation of tra ^–Flac mutants.