DNA Sequence and Units of Transcription of the Conjugative Transfer Gene Complex (trs) of Staphylococcus aureus Plasmid pGO1

Keywords: Ap, ampicillin, Tc, tetracycline, Nov, novobiocin, Rif, rifampin, Cm, chloramphenicol, Gm, gentamicin, Tp, trimethoprim, Qam, quaternary ammonium compounds, Trs, staphylococcal conjugative transfer

The conjugative transfer genes of 52-kb staphylococcal R plasmid pGO1 were localized to a single Bg111 restriction fragment and cloned in Escherichia coli. Sequence analysis of the 13,612-base transfer region, designated trs, identified 14 intact open reading frames (ORFs), 13 of which were transcribed in the same direction. Each ORF identified was preceded by a typical staphylococcal ribosomal binding sequence, and 10 of the 14 proteins predicted to be encoded by these ORFs were seen when an E. coli in vitro transcription- translation system was used. Functional transcription units were identified in a Staphylococcus aureus host by complementation ofTn917 inserts that abolished transfer and by Northern (RNA) blot analysis of pGO1 mRNA transcripts. These studies identified three complementation groups (trsA through trsC, trsD through trsK, and trsL-trsM) and four mRNA transcripts (trXA through trsC [1.8 kb], trsA-trsB [1.3 kb], trsL-trsM [1.5 kb], and trsN [400 bases]). No definite mRNA transcript was seen for the largest complementation group, trsD through trsK (10 kb). Comparison of predicted trs-encoded amino acid sequences to those in the data base showed 20%o identity of trsK to three related genes necessary for conjugative transfer of plasmids in gram-negative species and 32% identity of trsC to a gene required for conjugative mobilization of plasmid pC221 from staphylococci. 

Conjugative plasmids were first found in staphylococci in 1979 in association with outbreaks of gentamicin-resistant staphylococcal infections in hospitals in the United States (3, 11, 17). They were found in both coagulase-positive and coagulase-negative staphylococci and were shown to medi- ate interspecies transfer of gentamicin resistance (3,11,28). The widespread dissemination in U.S. hospitals of staphylococci carrying conjugative plasmids paralleled a dramatic increase in multiresistant staphylococci that caused serious nosocomial infections (4,38). 
Staphylococcal conjugative plasmids are large, range in size from 40 to 60 kb, and transfer at a low frequency (104 to 106 transconjugants per donor) (3, 11). Transfer does not occur in broth but requires a solid substrate, such as nitrocellulose (3,11). All of the genes necessary for transfer are plasmid encoded (43), as are a variety of antimicrobial resistance genes. Genes that encode resistance to aminoglycosides, ethidium bromide, quaternary ammonium com- pounds, trimethoprim, and penicillin have all been found on conjugative plasmids in staphylococci (1,4,13,14). Conjugative plasmids are also capable of mobilizing smaller, nonconjugative plasmids carrying resistance to chloram- phenicol and tetracycline (11). The 52-kb conjugative plasmid used in this study, pGOl, encodes resistance to amino- glycosides, trimethoprim, and quaternary ammonium compounds and is virtually identical to other conjugative staphylococcal plasmids isolated in the United States and other countries (2,14). Therefore, this plasmid serves as a model for study of the genetic organization of the transfer region of staphylococcal conjugative plasmids. 
The region of pGO1 containing the transfer genes has been previously identified by transposon insertional inactivation and subcloning (43). To determine the genetic organization and, ultimately, the function of individual genes in the transfer region of pGO1, we sequenced the 13,612-base transfer region (designated trs, for transfer, staphylococci), determined the number of open reading frames (ORFs) encoded by this region, and identified functional transcription units. These data constitute the first sequence and transcriptional analysis of an entire gram-positive conjugative transfer region and should provide a basis for comparison to other well-identified transfer systems. 

MATERIALS AND METHODS

Bacterial strains and plasmids. The Staphylococcus aureus and Escherichia coli strains and plasmids used in this study are listed in Table 1. Recombinant plasmids were generated in E. coli HB101 or XL-1 Blue (8,9). Recombinant plasmids constructed in E. coli were given pGO designations. When pSK265 carrying chloramphenicol resistance was added as a staphylococcal replicon to create a shuttle plasmid, the letter C followed the pGO designation. 
Materials and media. Mueller-Hinton agar (BBL Microbiology Systems, Cockeysville, Md.) was used for culture of both E. coli and S. aureus strains. Lennox L base (GIBCO- Bethesda Research Laboratories, Inc. [BRL], Gaithersburg, Md.) broth supplemented with 0.2% glucose was used exclusively for culture of E. coli strains, while brain heart infusion (BHI) broth (Difco, Detroit, Mich.) or Trypticase soy broth (BBL) was used exclusively for culture of S. aureus strains. The antibiotic concentrations used were as follows: chloramphenicol at 10 pg/ml for initial selection of S. aureus clones after electroporation and transduction and at 40 ,ug/ml for shuttle construction in E. coli and maintenance of S. aureus shuttle plasmids; gentamicin at 5 Pg/ml and erythromycin at 10 ,ug/ml for initial selection of S. aureus transductants; erythromycin at 20 ,ug/ml for plasmid maintenance; novobiocin and rifampin each at 10 pug/ml for E. coli strains HB101 F-hsdS20 (rB mB) recA13 aral4prA2 lacYI galK2 rspL20 xyl-15 mitl-supE44 X- XL-1 Blue recA-(recAl lac endAI gyrA96 thi hsdR17 supE44 reLUI [F' proAB lac-1 lacAM15 TnlO]) Restriction-deficient derivative of 8325-4 14.4-kb BglII B fragment of pGO1 subcloned into BglII site of pOP203(A2+); 43 Gentamicin resistance gene probe; 12 14.4-kb BglII B fragment of pGO1 subcloned into BamHI site of pBR322; 43 7.0-kb BglII-AvaI subclone of pGO200 produced by AvaI digestion of pGO200 and religation; 43 9.3-kb HindII-BglII subclone of pGO200 produced by HindIII digestion of pGO200 and religation; 43 Deletion derivative of pGO200; 43 4.4-kb HindIII E fragment of pGO1 cloned onto pBR322; 43 6.2-kb EcoRI C fragment of pGO1 subcloned into EcoRI site of pBluescriptIIKS(+); this study 2.4-kb EcoRI-HincII of pGO200 subcloned into EcoRI-HincII site of pBluescriptIIKS(+); this study 766-base BglII-EcoRI fragment of pGO53 subcloned into BamHI- EcoRI site of pBluescriptIIKS(+); this study 2.2-kb EcoRI-HindIII fragment of pGO200 subcloned into EcoRI- HindIII site of pBluescriptIIKS(+); this study 3.7-kb EcoRI-SphI fragment of pGO200 subcloned into EcoRI-SphI site of pUC19; this study 5.2-kb NcoI-SmaI subclone of pGO171 produced by NcoI-SmaI digestion of pGO171, treatment with Klenow fragment, and blunt- end ligation; this study 5.0-kb AccI-SmaI subclone of pGO171 produced by AccI-SmaI digestion of pGO171, treatment with Klenow fragment, and blunt- end ligation; this study 4.7-kb HphI-EcoRI fragment of pGO171 blunt ended by treatment with mung bean nuclease and ligated into SmaI site of pUC19; this study 3.4-kb HindIII-SphI fragment of pGO210 subcloned into HindIII-SphI site of pBR322; this study selection of plasmids transferred between S. aureus pairs by conjugation; ampicillin at 50 ,ug/ml for selection of E. coli HB101, XL-1 Blue, and TB1 clones; tetracycline at 12.5 pug/ml plus ampicillin for selection of all XL-1 Blue clones. 
The pBluescriptIIKS(+) cloning vector and the XL1-Blue host strain were from Stratagene (La Jolla, Calif.), and the pUC19 cloning vector was from New England BioLabs (Beverly, Mass.). All chemicals and antibiotics were from Sigma Chemical Co. ( St. Louis, Mo.). Restriction endonu- cleases and other enzymes involved in DNA manipulations were obtained from BRL. Agarose and acrylamide were from International Biotechnologies Inc. (New Haven, Conn.). DNA sequencing was done with Sequenase Version 2.0 (United States Biochemical, Cleveland, Ohio) and [cx-35S]dATP or [(a-33P]dATP (DuPont, NEN Research Prod- ucts, Boston, Mass.). DNA probes for Northern (RNA) analysis were generated by nick translation with [x-32P] dCTP and a kit from DuPont, NEN. VentR polymerase for polymerase chain reactions (PCRs) was from New England BioLabs. 
Cloning, transformation, and DNA manipulation. All re- striction endonuclease digestions, ligations, Klenow reac- tions, and electrophoresis were performed as recommended by the manufacturer. Transformation of recombinant plas- mids into E. coli was done by electroporation with a Gene Pulser (Bio-Rad, Richmond, Calif.) set at 200 fQ, 25 OF, and 2.5 kV. For transformation into S. aureus, the Gene Pulser was set at 400 fl, 25 OtF, and 2.3 kV. E. coli cells were prepared in accordance with the manufacturer's specifica- tions prior to electroporation; S. aureus cells were prepared in accordance with the method of Schenk and Laddaga (39). 
Staphylococcal plasmids were isolated for small-scale re- striction digestion by the cetyltrimethylammonium bromide extraction method of Townsend et al. (44). The alkaline minilysis technique described by Sambrook et al. (37) was used to isolate E. coli plasmid DNA for restriction digestion and subcloning into S. aureus. Plasmid DNA was purified from E. coli for sequence analysis by the Midi-prep proce- dure (Qiagen, Chatsworth, Calif.). 
Sequence analysis. Sequence analysis was performed by the Sanger dideoxy-chain termination method with double- stranded DNA as the template. Sequence reactions were run in accordance with the manufacturer's specifications for sequencing of double-stranded DNA with Sequenase 2.0, with the following exceptions: DNA was denatured in 0.2 M NaOH-0.2 mM EDTA for 5 min; annealing reactions were done at 370C for 20 min; termination reactions were done at 40'C for 5 min; and samples were heated at 950C for 2 min prior to being loaded onto gels. A 2.5-jud portion of each reaction mixture was loaded for 2-, 4-, and 6-h runs on a gel (14 by 30 cm) with an International Biotechnologies gel apparatus and a BRL model 4000 power supply at 87 V. Both strands of the 13.6-kb trs region were sequenced by subclon- ing of fragments (see Fig. 3) and by using sequentially synthesized oligomeric DNA primers to progress along cloned DNA. Sequences across restriction sites used for subcloning were confirmed on the intact BglII-B clone (pGO200). The trs DNA sequence was analyzed by the Genetics Computer Group (GCG; Madison, Wis.) program for potential ORFs and specific motifs. PCR generation of trs fragments. PCR was used to locate sites of Tn917 and Tn9171ac insertions more precisely than was possible by restriction mapping, and it was also used to generate DNA probes for Northern analysis of RNA tran- scripts. For transposon mapping, we synthesized oligonucle- otide primers that were complementary to either end of Tn9J7, on the basis of the published sequence (41), and to a nearby trs sequence. Oligonucleotides were chosen to gen- erate fragments between 200 and 600 bp long. The size of the fragment generated was estimated by gel electrophoresis with comparison to a 100-bp ladder (BRL). Sequence anal- ysis of several of the transposon-ts junctions confirmed that estimation of PCR fragment size located transposon inser- tions with an error of no greater than 50 bp. 
PCR generation of DNA probes for Northern analysis used oligonucleotide primers complementary to sequences of ORFs as determined by trs sequence analysis. Twentyeight cycles of PCR amplification were completed with VentR polymerase (New England BioLabs). Annealing reac- tions were done at 50C below the predicted melting temper- ature of the primers, and their template and extension reactions were done at 5YC above the melting temperature. 
E. coli S30 coupled transcription-translation studies. Clones of trs were analyzed for protein production by using an E. coli S30 coupled transcription-translation system (S30 ex- tract minus methionine [Promega, Madison, Wis.]). Protein products were labelled with [a-35S]methionine (Dupont, NEN) in accordance with the manufacturer's specifications, with the following modifications. The incubation period for all reactions was increased to 2 h. To maximize protein production, 15 Rg of DNA was used in each reaction with a cloned trs plasmid (pGO200, pGO203C, or pGO210) and 3.5 ,ug of DNA was used with vector plasmid. Reaction volumes of 50 p1l were precipitated with 200 ,lI of cold acetone and pelleted as described by the manufacturer. The protein pellet was suspended in 90 ,ul of sample buffer (0.0625 M Tris-HCl [pH 6.8], 2% sodium dodecyl sulfate, 10% glycerol, 0.002% bromophenol blue), 10 ,ul of 2-mercaptoethanol was added, samples were boiled for 2 min, and 50 ,u of each sample was loaded for separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (15% resolving and 5% stacking gel) as described by Laemmli (22). The gel was dried as recommended by the manufacturer, and labelled proteins were visualized by autoradiography with overnight exposure to Kodak X-OMAT AR film at -70°C. 
Northern analysis. Total cellular RNA was isolated from S. aureus pGO1/4220 as follows. A 150-ml volume of fresh BHI broth was added to a 250-ml overnight culture and incubated at 37°C with shaking for 1.5 h. Cells were pelleted by centrifugation at 10,000 x g for 10 min. The cell pellet was suspended in 5 ml of lysis buffer (20% sucrose, 20 mM Tris [pH 7.6], 10 mM EDTA, 50 mM NaCl), 1 ml of 1.5-mg/ml lysostaphin was added, and the mixture was incubated on ice for 1 h as described by Kornblum et al. (19). The lysate mixture was then centrifuged at 12,000 x g for 10 min at 4°C, the supernatant was discarded, and the pellet was thor- oughly suspended by vortexing in 20 ml of RNAzol B (Cinna/Biotecx Laboratories, Houston, Tex.). RNAzol B is a cocktail of guanidium thiocyanate and phenol formulated specifically for isolation of RNA. The isolation procedure was done as described by the manufacturer. Briefly, this consisted of extraction of RNA from the bacterial lysate with RNAzol and 0.1 volume of chloroform, followed by precip- itation in 1 volume of isopropanol and washing with 75% ethanol. The concentration of RNA was determined by measuring optical density at 260 and 280 nm. Samples were run on a 1.2% agarose minigel, stained with 1-,ug/ml ethidium bromide, and examined by UV light for the presence of two distinct rRNA bands. All laboratory ware was baked at 80°C overnight, and all water and buffers were treated with diethyl pyrocarbonate to eliminate potential RNase contamination. 
Northern blot procedures were done under the conditions described by Kornblum et al. (19). Briefly, RNA was separated by electrophoresis in 1.2% agarose containing formal- dehyde and transferred to nitrocellulose overnight in 20x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate). Nitrocellulose was baked for 2 h in a vacuum oven and prehybridized in Denhardt's solution for 4 h at 580C. Blots were then hybridized overnight at 580C with Denhardt's solution containing the appropriate [a-32P]dCTP-labelled probes, washed and exposed to X-ray film at -70'C for a time appropriate for optimum visualization of signals. Radiolabelled DNA probes were generated by nick translation of the PCR products of trsA, trsB, trsC, trsD, trsH, trsI, trsJ, trsL, trsM, and trsN. The clone pGO187, containing the 766-bp EcoRI-BglII fragment of trs that included trsN, the intervening promoter sequences between trsN and trsA, the first 244 bases of trsA, and the vector, was also used as a probe. Transcript sizes were determined for each trs probe by comparison to RNA ladders (high-and low-molecularweight RNA markers from BRL). Total cellular mRNA from mating mixtures of pGO1/4220 (donor) and 4220 (recipient) was also analyzed by Northern blot analysis to determine any induction of trs genes during the mating process. Briefly, after 4 h of filter mating, the bacteria were swabbed off the filter membrane, suspended in 1 ml of saline in a 1.5-ml Eppendorf Microfuge tube, and pelleted by centrifugation. RNA was isolated as described above by using 1/20 volumes of solutions. 
Filter mating. Overnight BHI agar cultures of donor and recipient strains were resuspended in saline to a no. 3 McFarland standard, mixed in a 1:1 ratio (2 ml of donor to 2 ml of recipient), and forced through a syringe onto a 25-mmdiameter, 0.45-,um-pore-size nitrocellulose filter. The filter was placed on BHI agar, bacterium side up, and incubated at 37°C overnight. Cells were vortexed off filters in 1 ml of saline and plated on Mueller-Hinton agar containing appro- priate antibiotics with selection for donors, recipients, and transconjugates. For each mating involving complementation studies, pGO1 and transposon-inactivated pGO1 deriv- atives were run as positive and negative controls, respectively. 
Transposon insertion mutagenesis. Tn917 insertions were generated in restriction-deficient strain RN1030 and were described in a previous publication (43). P-Galactosidase fusions were generated for this study by using Tn9171ac as follows. The delivery vehicle for Tn9l71ac was plasmid pTV32ts, a temperature-sensitive replicon designed for cre- ation of P-galactosidase fusions in Bacillus subtilis (52), which encoded resistance to chloramphenicol on the deliv- ery vehicle and resistance to erythromycin on the transpo- son. The delivery plasmid was introduced into S. aureus RN450 by protoplast transformation by using previously described techniques (43), and pGO1 was introduced into the same strain by conjugation. The strain containing both plasmids was inoculated into BHI broth and grown overnight at the permissive temperature for pTV32ts replication (30°C). The strain was then taken through two cycles of 10:1 dilution and then incubated for 18 h in Mueller-Hinton broth plus 20 pg of erythromycin per ml at the nonpermis- sive temperature for replication (420C). The bacteria were plated on Mueller-Hinton agar with erythromycin at a dilu- tion that yielded single colonies, and individual colonies were then picked to agar containing chloramphenicol. Eryth- romycin-resistant, chloramphenicol-susceptible isolates were screened for the inability to transfer gentamicin (pGO1) and erythromycin (Tn9171ac) into recipients, indicating strains with a transposon insert that inactivates conjugative trans- fer. The screening technique involved inoculation of 0.1 ml of donor and recipient colonies directly onto filters with a micropipet as previously described (43). Those yielding no transconjugants by this technique were confirmed as transfer inactivated by forcing donors and recipients through filters with a syringe as described above. Approximately 5 to 10% of erythromycin-resistant, chloramphenicol-susceptible colonies were inactivated for conjugative transfer. Tn9171ac fusions oriented in the direction of transcription were localized to trsI, trsK, and trsL by PCR as described above. 
Transduction and complementation studies. S. aureus transducing phage (D11 was used to lyse strain 4220 contain- ing pGO203C pGO171C, pGO324C, pGO325C, pGO311C, pGO338C, and pGO349C (see Fig. 3). Phage lysates were then used to infect S. aureus RN1030 (recombination deficient ()11 lysogen) containing pGOl with a Tn917 insert that had inactivated its ability to transfer. All Tn917 inserts had been generated in the RN1030 strain to facilitate genetic complementation without recombination (43). However, Tn9171ac insertions had been generated in nonlysogenized, recombination-proficient strain RN450. To transduce plasmids into RN450, phage lysates were mixed with bacteria in a medium containing 8 mM sodium citrate as described by Kasatiya and Baldwin (18). Cells were selected on chloramphenicol and erythromycin as previously described (43), and appropriate transductants were confirmed by cetyltrimethylammonium bromide lysis and electrophoresis. 
Nucleotide sequence accession number. The nucleotide sequence reported here (trsA through trsN) has been sub- mitted to GenBank with accession number L11998. 

RESULTS

Sequence analysis. The 13,612-base sequence of S. aureus trs is shown in Fig. 1. Some of the sequence of the insertionlike element that is directly repeated at each end of trs is included. The true sequence of trs begins and ends with the first base from the end of this element (43). The sequence of IS431/257 has been determined at multiple different insertion sites, thus defining its ends (6,10). Fourteen intact ORFs were identified by GCG program analysis. These ORFs, designated trsA through trsN (shown schematically in Fig. 2), range in size from 189 to 2,019 bp. trsA to trsM have the same direction of transcription, while trsN is transcribed from the opposite strand. There is one partial ORF at the 3' end of the predominant coding strand, just beyond trsM, that extends for 150 bp before it is interrupted by the insertion element. Since it is unlikely to have a role in transfer, this ORF was not given a trs designation but is called ORF O' and will not be considered further in this report. A summary of trs ORFs is shown in Table 2. All ORFs had near- consensus ribosomal binding sites (AGGAGGA, between -8 and -12 from the translational start site), and four, trsA, trsD, trsL, and trsN, had near-consensus promoter regions (TATAAT at -10 and TTGACA at -35) as predicted by the GCG PAlTERN.DAT and SEARCH programs. These near- consensus ribosomal binding sites and promoter regions are similar to those previously identified for other staphylococ- cal genes (31). The putative promoter regions of trsA and trsL are strikingly similar, as shown in Fig. 3. The -35 regions of these predicted promoters also contain a five-base sequence, ACATG, that is directly repeated three times in succession. Similar five-base sequences are repeated three times from bases 414 to 428 on the same strand as trsA and from bases 429 to 414 on the opposite strand. The first triplet of five-base repeats has a T in place of the first A in the final repeat, and the triplet on the opposite strand has the first and third A's replaced by T's. The triplet repeats from bases 414 to 428 are in position to provide another -35 region and, together with a near-consensus -10 sequence at base 450, provide a possible tandem promoter for trsA. The triplet repeats on the opposite strand overlap those of the -35 sequence for the trisA tandem promoter and provide the predicted -35 sequence for the tisN promoter. Most of the predicted trs ORFs begin with an ATG translational start codon, except for two, trsA, which begins with TTG, and trsJ, which begins with GTG, both of which are known to be functional in S. aureus (31). The start codons for trsD and trsK overlap the end of the preceding ORF. The remaining trs ORFs have an average of 14 bases between the preceding stop codon and the following start codon, with two exceptions. trsB and trsC have 59 bases between them, and trsK and tisL have 75 bases between them. Sequence analysis of the regions between trsB and tisC, between trsK and trsL, preceding trsA, and following trsM identified sequences with dyad symmetry. The regions between trsB and trsC and following trsM were predicted by the GCG TERMINATOR search program to be rho-independent terminators, but the sequences showing dyad symmetry between trsK and trsL and 5' to trisA were not. The palindromes 5' to trsA and trsL are perfect 8-and 17-base inverted repeats, respectively. In addition, the entire palindrome 5' to trisA, including the intervening six bases between the repeats, is identical to the internal eight bases and the intervening sequence of the palindrome 5' to trsL, as shown in Fig. 3. No other terminator or repeated sequences were identified in trs. 
E. coli S30 coupled transcription-translation studies. By using various subclones of trs, 10 of the 14 predicted trs-encoded proteins were visualized by autoradiography. By comparing the labelled proteins in each lane to the vector control lane, trs-encoded proteins were identified, as shown in Fig. 4. Visualized proteins were approximately the pre- dicted sizes for tisA (38 kDa), tisB (12 kDa), tisC (16 kDa), tisD (28 kDa), trsE (78 kDa), tisF (50 kDa), trsG (38 kDa), trsL (34 kDa), and trsM (16 kDa). trsA and trsG were not visualized as separate bands, since they are of the same approximate molecular weight. The protein product of tisN was also labelled and visualized with this system by using either pGO187 or the PCR product of tisN with its promoter (40) but is not shown in Fig. 4. The band identified as trsL in lane D appears to migrate to the same place in the gel as a vector protein in lane A. However, we believe that the band in lane A is the Tet protein that migrates at a molecular weight of 36,000 (15). This band should be missing in lanes B, C, and D because the tet gene was interrupted by cloning into the HindIII and BamHI sites of the vector. Proteins corresponding to the predicted sizes of trsH, trsI, trsJ, and trsK were not clearly demonstrated, but a faint band corre- sponding to trsH may have been present. The predominate labelled proteins for vector pBR322 DNA (31.5 and 114 kDa) correspond to the predicted sizes of P-lactamase and its precursor form, respectively. The difference in the numbers of vector-encoded proteins seen in lane A with the vector alone versus the lanes containing clones is not clear, but the patterns shown were reproducible following multiple replications of the assay. 
Complementation studies. Tn917 or Tn9171ac inserts into trs that abolished transfer were precisely mapped by PCR, and inserts into specific ORFs were then targeted for com- plementation studies to determine transcriptional organization within tis. As shown in Fig. 5, transposon inserts were localized to ORFs B (pGO1-83), C (pGOl-230), D (pGO1-106), E (pGO1-94), F (pGO1-15 and pGO1-86), I (pGO1-2P), K (pGO1-43), and L (pGO1-96). Various subclones of trs that contained successive deletions from the right end (beginning with trisA) were used for these experiments. These subclones are also shown in Fig. 5. At least 200 transcon- jugants were routinely seen if complementation of transfer genes was successful. If fewer than 10 transconjugantes were detected, the transposon insert was not considered to be fully complemented. Vector readthrough was felt to account for these results, since cloning of the fragment in the opposite orientation abolished complementation. 
pGO203C, containing the entire region upstream of trsA, trsA to trsH, and part of trsI, was able to complement inserts into trsB, trsC, trsD, trsE, and trsF, and plasmid transfer occurred at frequencies seen for pGO1 (10-5 to 10-7). pGO171C, however, with a deletion of the upstream region and translational start site of trsA, was unable to complement inserts into trsB and trsC but was able to complement inserts into trsD, trsE, and trsF. pGO324C, with a deletion of trsA and all of trsB, except for the last 25 bases, and pGO325C, with a deletion of trsA, tisB, and 90 bases of trsC, were both also able to complement inserts into trsD, tisE, and tisF. However, pGO338C, with a deletion of trsA, trsB, trsC, and the translational start site of trsD, was unable to complement any downstream inserts. pGO210C, containing the 3' terminus of trsI and all of tisJ, trsK, trsL, and trsM, was able to complement inserts into trsL but not trsK or the 3' terminus of trsI. pGO349C was able to complement an insert into trsL but not an insert into trsK. This subclone contained the left end of trs from the SphI site, deleting the 5' 240 bases of trsK but containing intact trsL and tisM and all sequences between trsK and trsL. Finally, pGO311, containing intact trsH and trsI, was unable to complement an insert into trsI. A summary and diagram of the results of the complementation studies are shown in Fig. 5. On the basis of these data, we concluded that three transcription units were identified by available transposon inserts. One contained trsA through trsC, another contained trsD through trsK, and a final one contained trsL and probably trsM, although there were no inserts into trsM to complement. Northern analysis. Two probes were used as controls for Northern analysis, a probe for the gene that encodes gen- tamicin resistance (aac-aph) and a probe for the gene that encodes staphylococcal protein A (45). The gentamicin resistance gene probe (pGO137 [12]) served as a control for the amount of message encoded by a constitutively ex- pressed gene on the same plasmid. The protein A gene probe FIG. 1. Complete nucleotide sequence of tirs (GenBank accession number L11998). Lowercase letters indicate insertion sequences that define the boundary of the trs sequence indicated by capital letters. The amino acid sequence of predicted tirs-encoded proteins is above the corresponding DNA sequence. SD, Shine-Dalgarno region. Translational start and stop are indicated for each ORF, and putative promoter regions are designated -35 and -10. Tn917 and Tnl971ac approximate insertion sites (+50 bases) are as follows: pGO1-83, 1,630 (trsB); pGO1-230, 2,100 (trsC); pGO1-106, 2,356 (trsD); pGO1-94, 3,219 (tsE); pGO1-15, 5,681 (trsF); pGO1-86, 5,924 (trsF); pGO1-2P, 9,337 (trsI); pGO1-43, 11,216 (trsK); pGO1-96, 12,770 (trsL). The exact deletion sites of pGO200 that resulted in pGO200A1 (at bases 2,367 and 12,363) are designated by the symbol A. 1AGAA TTAGCK I KGA AA KT1G8TS A K N AV 11401 TTTAGCAACTTATCGAVAGASTATTATTAGTATCACAACAATAGTTCAATCKA: 
E R S A A S V N D F 201 ATTTCTCTACCAGCTTGTGAAACGTTATCGAN 17 V K K K K G F F V S N 301 CTTTTTTCTTTTTACCAAAAAAGACTGAATTT _ -35_-35(P2). 401 ATACCACATTTACACATGACATGTCAITJ SD trA " D A 501 ATASAAMfaAIWSTTGTAA23NSATAACATA 28 N Y 0 5 R P G V K D 601 AAATTATCAATCAAGATTTGGTGTGAAAGATO 61 E D R E D K A K V L L 701 GAAGATAGAGAGGATAAACAAAAAGTTCTTTl 95 T F G N E T N Y E N 801 ACACTTTTGGTAATGAACCAATTACGAAATI 128 Y H 1 R 0 I K L A I 901 GTATCACAATCGACAAAAGCAATTACAATAT 161 E D L A L S L G L T A 1001 GAAGATTTAGCGTTAAGTTTAGAACTTACAG( 190 E N E N G K S E 1101 CTATAGAAAATGAAATTAATGGTAAGTCAGS 228 N R L G F E E F K N 1 1201 AATGAGGTTAGGATTTGAAGAATTTAAAAAC 261 N D P K V V A T E K G 1301 AACGACCCTAAAGTTGTGGCAACAGAAAAAA 295 A V K L S K E N K H 1401 AGGCAGTAAAATTATCCAAAGAATGAAACA 1 noNN K K F S L 1501 OTTGATTAATTIATTAAAAAATTCAGTTTI 31 P K I E 0 G L A 0 V 1601 SCCGAAAATTGAACAAGGTTTACAACACTAI 64 L P C I D D P N V It 11 1701 TTACCTGGAATTCATGATCCAATGCTAAAAA 98 P W V Y N L F Q * 1801 TTCCATGGGTATACAACCTTTTCCAAIMAC A E V KD I N L D N L 1901 CAAGAAGTTAAAGATATAATGCTCGATATAT 39 N L N A K E L A I V 2001 TAAATTTAAACGGTAAAAGATTAGCAATTG1 72 I T G F N V F I V 1 9 2101 AATAACTGGCTTTATGGTTTTTATACTAGGT 105 N I D T I K 0 S A R F 2201 ATGATTGATACCATAAAACAATCACAACGA1 7 K 1 K A N KI N E 0 2301 AACAATAAGCAAAATAAAAATAACGAACAAI 41 A G F Y K D Y V V T 2401 TAGCTGGTTTTTATAAAGATTACGTTGTTAI 7 o ND L L E E Y S S 2501 GCAAAATGATTTATTAGAAGAATAYTCATCI 107 Y I K H L Y R 0 V L 2601 TATATAAAACACCTTTACAGACAAGAATTAC 141 Q E K L A I G E N S 2701 TTCAAGAAAAGCTA C TAACA TGAI 173 V N R L N E K I N L 2801 ASTGIATAGACTACATGIAAGATAAACCTI 207 Y L N q F Y A L Y p 1 201 TACTTAAACCAGTTTTATGCTTTATATGAT 8 K I K Q E Q V T N K 3001 AAAGAAAAGCAAGAGCAAGTTACAAATAAO 41 F S W I E K K S N I 3101 TTTAGTTSGATAGAGAAAAAATCGCATATA( 75 S N L L KK N G N 1 3201 TATCAAATTTATTAAAGAAAAACGGTAATA 108 N K T A E Q N R T T 3301 AAACAAAACAGCAGAACAAATGCGTACTAC 141 D E K T S F N Y N I 3401 GATGAAAAAACAGGATTTATGTACATGTAT 175 T R L R L K T A T P 3501 TAACGAGATTAAACATTAAACACATACAC 208 0 0A N D T A T A A 3601 ACAACAAAATATOGATACAGCAACAGCAGO 241 K N T D S L V A V D
V S V D D V V D K 1 4701 ACGTAGGCGTTGATIATGTTGTTGATAAAI 608 Y G T 0 R A F I R I 4801 ATAC AACAC6ACATITAAGAAA 641 D V E P N Y V E 0 L 4901 GACGTAGAGCCGAATTATATTGAACAACTj SD trs 1 K I R N F N 5001 O8TAATCTCBIAATTA AMTTTTAP 31 D 1 8 E I R Y D L T 5101 GATATAGACUUTTAGATATGACTTAAC 65 T D N E Y N S F L 5201 ACACTGATAATGAATATAATTCCTTCCTA 98 D l I T V 1 N P A N 5301 GGACAATATAACTGTTATGCACCCAGCAU 131 1 N H L E 0 Y K L L 5401 ATAAATAATTTAGAACAGTATAAATTGTTI 165 K D D F F N K V N 5501 AAAAAGATGATTTTTTCCATAAAGTAAAT 190 L E E K RNK R E T 5601 ACTGIATGAAAAAAACGTAAAGAAAAA 231 F N I I N G K A A E 5701 TTTATGATTATAAA A 265 N V D A A V K G N 5801 GCAATGTTGTICAAGCAGTTAAAGGCATQ 29 
K A I E E Y R K R S I E A K K Y E S K 1 232 G E F P K P D K S K Y N G A S Y P A STAAAC3TATTGAAGAAATATCGAAAAGCGATCTATTGAGAAC6AAGAAATATGAGAGAA8ACT 7001 AGGAGAATTCCCTAAACCTGACAAATCAAATATAACGATCAATCTTATCCATCA 8 1 F N I K A I A T S K K K N K L V N A 265 K P V P L T A A N A A S A A D N A KI 'KAGTACTTTTAATATCAAAAACATTACTACAGATAAAAAAGA6AAATAAATTAGTCAT80CC 7101 AAACCAGTACCTTTAACTTGGAATAATAATAAAGACTAGGCAATAATGTCAAAM N K D N D N Y Y I N S A S T Y K K A V A N 298 5 V K P A N F A A P P P Y A N I N P AATAAAGATAATAATATGTATTATTTGAAATTCTAATAGCACTTATAAAGAG5CTGTTCAAM 7201 CCTCTG16AAACCCGGTAACTTTAGGAC8CACC6CACCGATG6TACA~TCATGT K L F I Y N S K N A A L T N Y D A I D K 332 N V K A I G V S S R E F 5 K A K T ATAAATTATTTATTTATAATTCAAAAAATCAAGCACTTACAAACTATGACCAACTTGATAV 7301 CAATGTAAAAGSCCTAGGAGTTAT6AATTCA6AGAGATVTTTAA~0M L E NE RK NRL T DTNMK VS3E NAK 3 KIL T I VI L FTL V LAAGC AN LTAAAAAACK ACAAATACGTAAAAAST0TAATSTA 7401 AAATTATTAACTATAGTTATTTTATTCACTTTGGTTCTGGCTGGTTTGCAT A LKANI N I N N V E K V T D N K S N V T 37 D T T K K R N S N I K A L I E Y A Y iTTGAAAAAGTAACATAAACATTTCTAATGTTGAAAAAGTCACTGATAATGAAAGTATGGTTA 7501 TAGATACTACAGAAUMAA6ACCA6TATGATAAAAGTCTAATTGATAT88VT, 8D FK D S IKEKA KEKKL T VSP S 70 S EK LAQKNaA E'LNaNNaS AN K kTTTC6AACTTTAAAGATAGTTTCATTGAAAAGGCTAAAGA6AAACTAACTGTTTCCCTTC 7601 ATCTGAAAAGTT TTGCA66CA AAAT0C6AATCAGCACAAAATAA a A N A F T K F F A A R Y D K S T L K 103 5 N D 8 8 L. Y S I K I S I T N K K ACAATAGCAATGAAATTTACAAAATTTTTTGACGCTAGATATAACG6AAA0CACTTTAGAA 7701 SAkTAATGATGATGAATTGATTTATTCTATTAAACTTTCAATTACAAACAAAAAAI S V F DK 6N K KDL T SA ST FE E 137 EE E KAKNaK ID0N V E L TNRNI rGTTTCTGTTTTTGATAAACAAAAAAATTTAACATCAGCTAACACTTTTGAGG 7801 CGGAAGASAGASAGTAAACAAAAGATAGATAATGTC"AGAAACT6CC00 ILSNRS EI E L N aK K K KKI' 55S 3 T IIL C EKp saA ND L ST V rcACTTTCTCATAGTGAAATAGAATTAAATCAAGAATTAGAAAAGAAOWAAOAiAOMi 7901 CACTTTAATTCTTATTA6AAAACCTTCACAAGCAATGQACTTAAGCATGTTTTl TYV A FL SI VL SN I T L AE NP G 36 L NV SAGF LTNWA V6N L V EL ,TTTATGTAGCATTTTTAAGCATTGTTTTATCAAATATAACACTTGGCGC6AGAAATCCAGG 8001 CAATT6AATTA6AGTGGCTTTTTAACTTGGGC6ATTGGTCATTTATCMT~T I L T QL I VA V IC AA0V WI V LKK 70 P IL L EK D D FAPK V S K TI FCTIAACAG CCTAATTATTACTGTTAATATCTATACTGATATTTGATAGTMTTTAAAAAA 8101 ATCCTATATTGTT6AGAAAAAACGATTTTCAATTTA6AAGTAT~CT A0 F R A V A N V I A A V A V GA6 A I V W I V 103 5 K V A T 8 P A R K A E N I A Y ITTTAGAGOCATTAAAATAATT rAACTAATGTASCTGTTGGGGACACACTTGTTTAATTGA 8201 TGATG6AAGTATTATTGCTACTAACCC6ACTAGAMAGA6AC 6TATAGCTA bo-irak0Land eminator.. SD.In& 98 g S VE 136 L NL T S3K V E8S I RK A K N 6AOCTACATTCTAATA6ATGTAC.TTTTAGTTVTAAA6M TGAATA6TATAIICTGTTG0G 8301 6A6TTGTGACTTACTTCTAAAATAGAAAGCAGTATAAGAA6AGGCTTTTMAAMA K K K V K K N I P R N V D A S Y K I F L 170 R K L S D W L V A N L S R N F TI A~kAAGAAAAAATTAMAAGAATATIAATACCTCGTAATGTAGACSCTAGTTATAAAATATTCT 8401 CTAGAGAATTAAGTG4TTGGTTAGTAAMVTTACTTATCACGTCATTTCATJ PF L L F L T A F A V T F L N G L L N L 203 V S S P T L N N V Y N R K N N I K A CCTTTTTTACTATTTCTAACAATAACATTT80TGTAACGTTTVTTATGGGCTTACVTTATTT 8001 AGTATCT6AGTCTACATTAAACAT80TTTACAACAGAGAAATAATCATTAMM AL T I YAL L T I R PI TKEN I R 236 SKEaKEV K TKELK NKPDSK3EDK TAAVTTTAACAATATATAACTTACTAACAATTAGACCAATTAGTACAA6AAAAAATATAAA 8601 GATAAACAAGAAAATAAAAACAGAAATAAAAAACAAATTTAAAATGAAAGATG8U R a K V Y F Y K S K E A L A 0 0 D 270 A L V T 5 I E K E I A Y T N P P KI * -1 80 trDN N N F K K 8701 AAGGATTAATTACTS6TATTGAAAAAAAATAGGATACAACTT6CAC6TAAA KASaCAAAATTACTTCjATAMAATAAAIMIBTTASATGAIATGATATTIMAMW 303 S I AAK RT LKEI AN0T L Y KK L I I N R Y I K A S SA N A S s ia E S S L 8 801 AATAAAISCGAAACGAACATTAGAA6ATCGCACAAACATTATATGAGMMAAAI AATTATAAAT6AATAT6AAAAA6CACTAATCAAAGTTTICAAGAAGATTCACTA 336 K E N I L K N I S Y L K K T K N S Y I S A A L N I T A V N T K L L T K 8901B" AAAGAAATATTATTAGAATAATTAAACTATTTAAAAGAAA6TAACAAAMATTAM SAGTTATATCAGTGG6AGTTTAMATATTACTGGTGTAAATACAGA6TATTAACGAAGTVTTA 370 K 8 N V A I I I T A N 8 P N K V D IN I L Y EP N I K TIT EP V N IA0N 9001 T6AAAGATCATTACGCAATATTAATAACTGGAAATATAATTTAACAAAGTAG 'CAAACATTCTTTACGACGAACC66AATGATAAAACAATTACAGAACCAATAAATATACAAAAT 403 A N N P N 8 K E N V K T T T I K I A K N N N N K P T N N A a I I A A N I P 1 910 GGCCATGAAATTTTATGATAATAGAA6CAATATGAAACGACTACTATAGAAATCS SGAAAAATAATAAIAATAAACCAACAAAACATAGCCAATTAATAGCTAATCGTATATTGTTTC 436 N A P K A I I N K a K T S K K T I P K N V I P P 8 K K I T K N T V A Q I S N A 9201 AACGGATTTAAAGCTTTGTTAAATAAACAAAAAACTTCCGA GACAAA TTCC AACTTATATTTTCTTTTCGGAAAAGATAACAGAAAATACTTAT6SCCAATTAGATATGTC 470 K K T T P P K N Y T K K T I I K A A I N N A P 5 N Y A I K T N P I V a V K 0301 CAAAAAGAAACACC6 ATAAATACAAAAAAAACATTATTAAAAAGCJ AAGCATTACAAC6AACATTTTCACATTATGATTTAGAAACIAAACCTATATATCAAACAGAA 503 1 K K V K A I A T P A T N A 8 1 1 E T N D VY N L SN SD UK N I NA FLK 9401 ACTT6AAAAAGTTA6AAGGTTTASCCCC0ACT TAC ATTATG 'TTATAACGACTATCATCTTAGTAATyM~AA~iM85ATTTIAWTThI3SCTTTTTAAA 536 Y T K N S I I A C I I I' KG N I I K S F a K I T K V I S T D 5 V S I Yo 9901 TATATTACTAAAAATAATATATTAACTTATTTATTATTAGAAAGGACAYTTACI ATGAATCTTTTCAAAAATTAAC6AAAGTATTAGATACAGATTCTGTTGATAGATTTTATCCT 570 5 K A K K 8 5 8 8 P N T I N K N KN V IK NILVLVV YPA0S V K GAATI 9601 TTAGTAAAAAAAAATGATATTCATTTATAAATACCATTTAATMTC CGAAAAATTATATTAAAAATTTATTAGTAATTGACTACCCTCAATCAGTAAAAGGGSCSCTACT 603 K V A K E K V S T N N I A K C P AC T K P I N P A N I E N N S H I N N S I K 9701 AAAAATAACAAAAATATCAACTAACAATATAACAAAATACCCTACTI ACTAAATTTATTAGACCAACAAATATAGAAAGAATGATTGACCATTTAAACAACTCAATCAA 636 K T N N A C K P T I P K K I I K K S 8 N A T I K N E I K S S K K a I N K P 1 9801 AAATACAATAACGGTTATAAATTTACTATTCCTAAAAAATTAVTAAA AACGAAAATSCAACAAVTAAAAAGAAAATTGAAASTAGTAAAAAACAACTTGATAAATTTTTA 670 K I K A F K S K K S A K S F 8 C K T I N A 5 5 1 K K I a A I K K D V K N 1 1 9901 AAAAATTAAAAGGCTTTAAATCAAAAAAAAGTGGAAATCATTTAATTGATM ~CACATTAAATGGCGATTCATACGAAAAAGATACAAOCSCTAGAAAAAGATATTAAGCATACTT 1 I 80 SW N K K A F I T A N I A N RE S PN T V'L PLN RNNFIS A FT 10001 0000&MTAA AA[NAAAATATTCATTTTAACTGATATTATTTAAC1 ACAATACQAAGATCATTTCATACANTTCTTCCTTTAAATAGAAACTTCTTATCAGCATTTAC 30 1 5 N T I N K Q K I F 1 5 1 s a I F rN F 8 8 S E I I 8 1 1 P N T S V F A N 10101 CTAAGAA6TACTAGCAATAAGCAAAAGATATTTAATGATATATC-ACA TMTTl rTTATGTTTAATGATTCAGAATTAVTTATTTAACACCTAATACATCAGTMTTTGAATAATC 64 D K N T V F F V I A V I A T I K K K K T I N K N N T I A T S A V A K 5 1 10201 CTGATAAAAATACAGTATTTVTTATAATAGGTSG6TAATAW-ACAATTATTTGAT kCAAAGAAAAAACTTTAAATAAAAATATGACTATTATCGATACAAATSGTATAAATAAATCTA 97 K N a S Y K A K H A I H A I A K F S N a F I 8 P K 8 E F 5 1 T K Y Y G 10301 AAAAAATCAAGACTATAAAATTSCTAAACATGGTTCTCATGAAAC6ACTAAAT7 TICTATAAAACAATTTATCATTAATKCCCAAGATAAATTTTCATATATAACTAAATATTACGG 130 K K N S T N Y V V N S
28ATATAA1TACAAATAAAAAAATAA0CASCAA6IATITI~h~3 43 A N H A V I C I S N V N N 5 1 A T I V A K N N N L E Y I N V D 1 C N I 1 11501 ATAGGTAACCACGCAAITATITTTATGTTAGGAAT GTAATAI AATAA GATAC.AAC ACATTGATAAAAAAACATAACCACTIAAAATATATCATCAATATTGATTI1AIMAT~AATC 377 5 5 E S 8 S S K S S N S K I S N K EL PPKEV K DL N0K K ASVP FN I I N 11601 CAAGTAATAAAAAOTCTTCTAOTOGTAASTACAATAAATCATCNA1AÑ TGAAATIT1ITTAAG1AATTTACAAAASAAACATT AITTTTVCATATCATTTAC 410 5 N N a a S K A I I T K N N N P K K KS K A LK L SL ND YVVL K V P LK 11701 AAGTAATATGCAACAGAAAIAGACTATICTTATAACTAAAAATAGAAATAITA AAAA CAACCCAAATTATCTTTAATGATTVATAIAITAAAAAGTTAATCCATTAAA 643 K T Y I A P a N K V K N K K N S S N P K N K 5 1 K I A P K A N K Y I P K P K 11801 AAACGTATATAGCACCTCAAAACGAATACAAACGTAAAAANAAACCAGTCIAT 1T1AAAAATATTGAGAAAATAAAAATAGATTICAAAGACCATGAATATITYACTAAA~CCAAA 477 K N K a a K I K E I K K E a N a K 1 8 A a Y P 8 K Y Y N N K P K V I S K E 11901 AGAAAAAATAAACAACAAAAACTAGAAGAATAATA.AASAAAAAAAN ATACATIATCACAATACACAITATATAATATATATCGCAATAAATTGAMAGTTTTATCAAA 510 V K K S K D K N S N K S K K S K N I T P E K I K K I V R N K I N a V A T S Y 1 1200 AAIAAGASAAAAAAGAACAATCAAGAAGAAAI AAArAACGAACNAU AACTTTTGAAAM TTGAAAGAGATTGTAAAACATGAATTGAAAICMAAACAGACAGIAVTTAT 543 K a A K *-35 K Y I N N I I I S P 1 8 I A V I S I 5 12101 MACAACTAAAIMAAAAAAATITTACATTACAI08MAIACATGTAATUC kAAGTACATTAGAAAAT11MTACTTASIITTGTT80TATTGCAA1ATAITAAG0TTMTATTC 4 1 K D A K K I T K S I A S A N N N IK I A A A S K A E N N S K V Y K N I Y N A 12201 AATTAAAGAACCATTAAAAAACIAACCGAAAAATATIaCAAACAAIATGAGA9
OCAAATAAAAAAAAACAACAAATAAAr.ATATGAAAMATCTTTACAAT 37 F P 1 W 1 1 5 K I I I S P N S I S S S S P N K K 1371CITI0TAIASAAIMT..TOOCO-OS__ttOO tttKtO90 
S K K T I K K K K K Y E K 1 12301 ITCITTATATGGTTATTAGAAAAAATACTTATTGATCCAATAATAVTTTC OAGATASCTTTMAACAAAAAAAAAGATA AAMAACATIAAAAAAAAAATGAAGAACT 71 IA I C VP APFK N va V N K E N L Y K E A K N N K I N K I A P N P K N

CAATAGCAATATGTTGTATAI1CVTTAAAATA0T1CAAGAITAAAAC8AAM AGAACAATTATATAAAGAAAAATAAGCAAGAATAAAATAAGAGASATAGCTTTCAAIITTTGAAAT

F E K M A K G G A L E G K G K D rTTTGAAAAAAACGCCG ASGGTGCGCATTAGAAGGTAAGGTAAGG kTTGTAGCT I E E :AATTGAA v a A S E S E W C R G A M A T K tTCTGAGTCAGAATCGGGAAGAGGGGCAAATGCAACAAA E G L D K C A K N L Y D L AAGGACTTGATAAASGTGCAAAAAATTTATATGACTTA S N D P D D L N S N W V P CTAGTAATGACCCAGATGACTTAAATAGTAACTGGGTAC S G G G S D C K G F D F K a T W Y V H Q R R K E I G CACACTGTACTTCCTATGTACATCAACCTACAAAAAAAATTGCC O G W E V C S K P K A G R C A CGCAA M TGGAAGTAGGTAGTAAGCCGCAAAGCTGCCGTGGCG V E K V K K D G C I V V S E A TTCGAAAAAAGTTAAAAAAGACGGTGAIATTGTAGTTAGTGAGGC R N F I D K -SO SM N K CAACGTATGCAATTTATGTATAATG 0GIGTAAIAAAWGAA -; Q T iO K S E K I AATCAAAITCATAAAAATA E D Y K E E K T V TGAAGAAAAAACAG N K D I L ATGAAAGATATATT Y D V -E K " =D Y-~R S Y" R YG C K ---IE L T GAGAAAAACTCTGACTACTCAATGAGATATGSGAATTGAATTAA L V S N N E * So I "N AATTTATAAGTATGAATCAAGAAISAAAGAOiAATACTTAI8 A K K K K O N a Y N E I S D E TTACAAAAA AAAAA TGGATATATGGAAATTTGCAATGAA K E P O E Y D E K Y K N f S T Y GAAAGAACCACAAGAATATGACGAAAAATATAAAAATTTTAGCACAT K D Q F N N I K K I F K E N K I AAAGATCAATTTAACAACATTAAAAAATATTTAAAGAAAACAAGAT 'K I L N L K V T D K V T I K IATAAAATTTTAAATCAATTGAAAGTAACAGATAAAGTTACTATAAIA I L P K E K T Y C f Y K E G It A VTATATTACCTAAAGAAAAAACTTATGGATTTTAAAAGAGGGCAGCG K I S It E L a N D G V I H I G R 'AAAAITTCACGAGAATTAGGGIAACGGTGTTATTCATATCGGTAG I F K G K K F Y K V S A T I N K IITTTTAAAaIATAAAAAGTTTTATAAAGTTTCAGCGACTATTAACAA L H E F L F E N D I T D L T Q K VTTGIACGAATTTTTATTTGAAAATGATATTACAGATTTAACAUAA ____ _D,__S A ,L, 0 E D N N D K Y K ----------S .....A-K -NNSKIKTAA R T D S R Y I T E D E AGAACTGATTCAAGATATATCACAGAAGATGAA T I I S L I A P S K I TAACTAATAACAVCTTGATTAATCCTTCTAAAA I A V Y K S I L TA V NATUATGTTTATAAGTATATTACAAAATGT N F E V K G K I I A D '1TGTTTGAAGTGAAAGGTAAAATTATTC1AGAT D I E L D L L E AACATTGAATTAGATTTGTTAG D E G L K S T AAGATGAAGGCTTAAAGTCTAC Q V Q K N K I TCAAGTACAAAAAAACAMAATT W E K Y L N C I kTGGGAAAAATATTTGAATGGTA f K N K E S I 0 'TTAAAAATAAAGAAAGTATACA K C F Y C C T MAGGTTTTTATGGTTGTACA L C E S K T T K kTTATGTGAAAGTAAGACAACAA L Q F S F D * TTATTCTCAKKVLNS K N" E I A NKKEE CED VLIVIAAII~~~~C TAIAIN TCTCGTAIA IV L I Y L A Q I L A Y A L N T 1TAGTATTIATATATTAACSCCAATATTAGCATATGCTTTAAATACA 111016 E5D1KTE TASGCGAAOS N IGAAACGA K N 6606610G E E R S D I K K SD la T T L L G E L E rAGAAAmA §TGATAASAAICACACTTTTAGGCGAATTAGA r K C P N R N I F V V C G P G S kCTAAATGCCCTAATAGAATATATTTGTGGTAGGAGGGCCAGGTTCC I V V T D P S G E V Y E K T S N NATAGTTGTAACTGACCCAGTGGCGSAAGTTTAIGAAAAAACATCTA L A S D R Q N F F D Y I D K D S X885 E G VyI NP__ ... N F A N L .I L DK P A R ESSKKEK GAATGGGATAAAAACAAG E Q Q N E K AAACACAAATCACM i E K
.__. _._. FIG. 2. Schematic representation of predicted trs-encoded ORFs, designated A to N, and units of transcription identified by complementation and Northern blot procedures. The entire pGO200 subclone of pGO1 is shown, including the restriction endonuclease cleavage sites used to create subclones. The region corresponding to the DNA sequence in Fig. 1 is represented by the line between the two dark boxes above pGO200. Lines with arrows above pGO200 indicate direction and units of transcription determined by complementation and Northern blot analysis. The top solid arrows repre- sent the trsA through trsC, trsD through trsK, and trsL-trsM transcripts identified by complementation. The arrows directly below this represent the transcripts identified by Northern blot analysis and are as follows: the first solid arrow represents the 1,300-base trsA-trsB transcript seen with pGO187 as a probe, and the broken line above trsC represents the 1,800-base trsA through trsC transcript seen with trsB as a probe; the broken line above trsD through trsK represents the predicted-size transcript based on DNA sequence analysis but not identified by Northern blot analysis; the final solid line represents the 1,500-base trsL-trsM transcript iden- tified with trsL and trsM as probes. Subclone pGO187 is also indicated. Restriction enzyme cleavage sites are abbreviated as follows, with the numbers in parentheses corresponding to nucle- otide sequence numbers in Fig. 1: B, BglII (1, 14,409); E, EcoRI (766, 7,005); A, AvaI (1,234, 6,825); Nc, NcoI (1,804); Ac, AccI (1,977); Hp, HphI (2,291); Hc, HincII (3,169, 6,533); H, HindIII (9,162, 9,211); Sp, SphI (10,716); X, XbaI (13,376). trsD and trsK are represented below the other ORFs, since their translational start sites overlap the translational stop sites of the preceding ORFs. The hatched boxes at the ends of pGO200 represent the insertion-like sequences that border trs. ,,2,AgaTTTT. .gcATTACATGACATGACATGTAATgCaAoatctGTTATAcTTGtATT ... .gAAgTAG .AGGTGG.TTG. .A&O I2193 3' 5 ' .AagTTTTatoaATTACATGACATGACATGTAATaCtAt .... GTTATAaTTG.ATTtatcAAA.TAGaaoAGGTGGOTTGtaAITTG .2 3' FIG. 3. Comparison of sequences 5' to the predicted translational start sites for trsL and trsA (promoter 1). Capital letters indicate identical bases, lowercase letters indicate nonmatching bases, and ellipses indicate gaps introduced to maximize similarities between the two sequences. Predicted promoter regions are marked -35 and -10. SD, Shine-Dalgarno region. The numbers at the beginning and end correspond to the nucleotide sequence numbers in Fig. 1. The arrows indicate areas of dyad symmetry. The translational start sites for trsL and trsA are in boldface type. served as a control for the amount of message present on the blot from a well-expressed chromosomally encoded gene. Both of these probes identified transcripts of the appropriate sizes (1,900 bases for the gentamicin resistance gene and 1,500 bp for the protein A gene [45]). As is evident from the Northern blot (Fig. 6), the highest level of message was detected with the protein A gene probe and the next highest level was detected with the gentamicin resistance gene probe. 
Figure 6 shows the transcripts most easily seen by North- ern blot analysis. The analysis was performed on eight separate occasions, and the results shown in Fig. 6 and presented below were consistently observed. Furthermore, with our RNA isolation technique, there were few back- ground or rRNA signals so that we could attribute even faint bands to hybridizing mRNA species. The trsN probe hybrid- ized to one abundant transcript of approximately 400 bases. Probe pGO187, containing the trsN gene and the first 244 bases of trsA, hybridized to two easily identified transcripts of approximately 1,280 and 400 bases, corresponding in size to a transcript containing trsA and trsB and one containing trsN. A band of approximately 1,800 bases was also seen with pGO187 as a probe but is not well visualized in Fig. 6. When trsA alone was used as a probe, faint bands of 1,280 and 1,800 bases were seen (data not shown). The trsN transcript was longer than the trsN ORF, presumably be- Autoradiogram of E. coli S30 coupled transcription- translation studies. Lanes: A to D, labelled proteins produced by specific DNA used for the reaction; A, pBR322; B, pGO203C; C, pGO200; D, pGO210. Rainbow molecular weight markers (Amersham) were used to size proteins, and the migration of these bands is indicated by dashes and the corresponding molecular weights (MW). Arrows with letters indicate the trs-encoded proteins corre- sponding to the protein bands visualized. Arrows with asterisks indicate the 13-lactamase protein (31.5 kDa) and its precursor form (114 kDa). The band above trsE (e) in lanes B and C did not correspond to any of the predicted trs-encoded proteins but may be a vector-encoded protein that is visualized faintly in lane A. A trsD-encoded protein (d in lanes B and C) was revealed by Western immunoblotting of the proteins produced in lanes B and C by using polyclonal antisera generated against the purified trsD gene product. cause of run-off transcription due to the absence of termina- tor sequences. The trsB probe also identified transcripts of 1,800 and 1,280 bases, no 400-base transcript, and a possible transcript of 500 bases. A weak signal of approximately 1,800 bases was barely visible when the trsC probe was used. The trsD, trsH, trsI, and trsJ probes did not identify any specific transcript but demonstrated a smearing down the lane which may suggest the degradation of a single large transcript recognized by these probes (29). The trsL and trsM probes each identified a single transcript of approximately 1,500 bases that is easily visible in Fig. 6. Comparisons of the levels of the trs-encoded message between donor cells alone (pGOl/4220) and mating mixtures of donor and recipient cells (4220) did not reveal any increase in message in the mating mixture (data not shown). 
Comparisons of trs to other transfer sequences. The predicted amino acid sequences of trs ORFs were compared to the predicted sequences of other ORFs in the GenBank data base by using the GCG TFASTA program. trsK showed significant homology to traD (GenBank accession number M29254), one of the genes in the E. coli F plasmid conjugative transfer operon (49); virD4 (accession number X06045), a gene on the Agrobacterium tumefaciens Ti plasmid that is involved in the transfer of this plasmid into plants (32); and traG (accession number X54458), a gene on broad-host- range conjugative plasmids RP4 and R751 (53). trsK showed 22% identity and 46% similarity to traD with 23 gaps over 643 amino acids, 22% identity and 48% similarity to virD4 with 19 gaps over 581 amino acids and 20.8% identity and 40.1% similarity to traG, with 10 gaps over 389 amino acids. Recently, Lessl et al. (24) compared the amino acid se- quences encoded by traD, virD4, and traG and found sequences shared among all three proteins, most of which we also found to be shared by trsK. Of the most interest, however, was the shared nucleotide triphosphate (NTP)binding motif seen among the protein products of gramnegative species that is highly similar in trsK. The type A consensus binding site (24) was not highly conserved for traG and virD4. However, trsK contained identical amino acid sequences over 9 of its 13 amino acids, 5IYvt1~ps _iEVYE (underlined capital letter indicate identity, and capital letters indicate similarity; bases 10,652 to 10,690 in Fig. 1), compared with the traG and virD4 partial NTP type A sequences. In contrast, the type B consensus NTP-binding motif was highly conserved among trsK, traD, traG, and virD4. The sequence YdELL12EW (bases 11,342 to 11,365 in Fig. 1) was identical or highly conserved over seven of eight amino acids with respect (i) to virD4 (indicated as described above), (ii) to traG over seven of eight amino acids, and (iii) to traD over six of eight amino acids. The contribution of the NTP-binding motif of the proteins in gram-negative species to their function is unknown. 
In addition, the predicted amino acid sequence related to that of mobB, of trsC was encoded on S. aureus plasmid pC221. We have previously shown that mobA and mobB, two overlapping ORFs, are essential for conjugative mobili- zation of this plasmid (33). trsC is 32% identical and 53% similar to the terminal 140 of the 233 amino acids of mobB of pC221 (GenBank accession number X02529 [34]), with four gaps. No additional significant homologies (>20% identity over 250% of the amino acid sequence) were found between predicted amino acid sequences of trs ORFs and sequences in the data base encoded by genes involved in conjugative transfer. In addition, because of the findings obtained with trsK, the predicted gene products of all trs ORFs were directly compared to the predicted amino acid sequences of all genes encoded in the Tral and Tra2 regions of RP4; no other significant homologies were found. Because of the similarities between gene products noted above, pC221 was introduced into pGO1-230, a transposon insert into trsC on pGOl that inactivates transfer, and no complementation was seen. Finally, the entire trs region clone (pGO200) did not complement a traG mutation in RP4 when both were intro- duced into E. coli (46). 

DISCUSSION

In an earlier study, the conjugative transfer region of staphylococcal plasmid pGO1 was localized by transposon insertion mutagenesis and by subcloning of a functional 14.4-kb BglII fragment on a separate staphylococcal replicon The trs ORF that corresponds to each insert is diagrammed above pGO200, and the subclones used for these experiments are diagrammed below pGO200. Only ORFs containing transposon inserts are included. All ORFs are shown in Fig. 2. Restriction enzyme abbreviations are as described in the legend to Fig. 2. (B) Subclones used for complementation are in the left-hand column. Tn917 and Tn91l7ac insertion numbers and the trs ORF into which each inserted (in parentheses) are listed at the top of each column. +, >200 transconjugates per mating; -,0 to 10 transconjugates per mating; ND, not determined since the corresponding region of DNA is not contained within the subclone. (43). In this study, we sequenced the entire fragment and determined its transcriptional organization. We designated the 13.6-kb region trs (transfer in staphylococci) to distin- guish it from the tra regions of plasmids from gram-negative species. We identified 14 intact ORFs that we feel are likely to be functional genes on the basis of the following data. (i) Each is preceded by a near-consensus ribosomal binding site, appropriately spaced from the translational start site. 
(ii) In an E. coli transcription-translation assay, proteins corresponding to the sizes predicted for 10 of the 14 trs ORFs were seen. (iii) Transposon insertions were localized to 8 of the 14 predicted ORFs, each abolishing transfer, and each could be complemented in trans for transfer by a clone containing the corresponding intact ORF. (iv) We identified transcripts corresponding in size to trsA through trsB, trsA through trsC, trsL, through trsM, and trsN. Thus, a combi- nation of analysis of gene products, complementation of inactivated genes, and identification of transcripts of the appropriate sizes demonstrated a probable functional role for all of the predicted ORFs except trsJ and trsK. The precise role for each of these ORFs in transfer will have to be defined in future studies. 
Additional evidence that two predicted trs-encoded pro- teins not seen well in the E. coli transcription-translation assay are produced came from Western blot (immunoblot) analysis. Polyclonal antisera previously generated against purified gene products of trsD and trsH identified both proteins with the predicted molecular weights (28) among total cellular proteins from S. aureus RN4220 containing pGO1 (28) and a band corresponding in size to TrsD among the proteins produced by E. coli transcription-translation of pGO203C and pGO200 (29). Proteins corresponding to the predicted product of trsI, trsJ, or trsK were not seen. This could be due to the inability of these staphylococcal genes to be transcribed and translated in an E. coli system, to poor resolution of the proteins during electrophoresis, or to the sensitivity of these proteins to degradation by cellular proteases. Although the S30 extract system is OmpT protease deficient, other proteases could be present in the extract that degrades these proteins. This hypothesis is supported by the observed degradation of the protein encoded by trsI by cellular proteases (28). 
We deduced transcriptional organization of the trs region from analysis of DNA sequence motifs, complementation for transfer of transposon inserts that abolished conjugation, and identification of mRNA transcripts on Northern blots. Three major complementation groups were identified. The 83(B) 230(C) 106(D) 94(E) 15(F) 86(F) 2P(H) 43(K) 96(L) first included trsA through trsC, a predicted transcript length of approximately 1,800 bases; the second included trsD through trsK, producing a 9,900-base transcript; and the third included trsL and trsM, a 1,500-base transcript. Allow- ing for two mismatches from the consensus E. coli -10 and -35 sequences (TATAAT and TTGACA) and by compari- son with known staphylococcal promoter sequences (31), near-consensus candidate promoter sequences were identi- fied 5' to trsA, trsD, and trsL, consistent with the comple- mentation data. Secondary structures containing areas of dyad symmetry following trsK and trsM may serve to terminate transcription at those sites. There is no sequence that is an obvious transcriptional terminator between trsC and trsD; in fact, the translational stop of trsC and the start of trsD overlap. Rather, there is an untranslated 58-base gap between trsB and trsC that contains a predicted rho-independent terminator. Some of the Northern blot data are consistent with these sequence motifs. Transcripts of 1,800 bases, consistent with transcription of trsA, trsB, and trsC, and a fainter band of 1,300 bases, equal to the predicted transcription of only trsA and trsB, were seen when trsB was used as a probe. A probe (pGO187) containing trsA showed a 1,300-base major transcript with a faint band that may represent the 1,800-base transcript. The 500-base transcript identified by the trsB probe could not be explained from the sequence or complementation data but may be the result of message degradation or processing. Probing with trsC re- vealed only a very faint band that may be the 1,800-base transcript. These data are consistent with transcription of trsA and trsB at some stages of conjugation, with tran- scription of trsA, trsB, and trsC occurring at other times. Transcription of trsC could, therefore, be regulated by antitermination. Instability or low abundance of transcripts containing trsC may explain the difficulty in demonstrating a transcript by using this probe. Since there is no predicted terminator sequence downstream of trsC, trsA through trsC may at times be transcribed as trsA through trsK. However, since a construct removing sequences 5' to trsA could complement transposon inserts downstream of trsD but not those in trsB or trsC, trsD through trsK transcription must predominate. No transcript corresponding to the large pre- dicted complementation group trsD through trsK was seen when tisD, trsH, trsI, or trsJ was used as a probe in any of the Northern blot procedures. This may have been due to degradation of this long message in the process of RNA preparation or the message's inherent instability or low abundance. 
We also identified regions that will be targeted for further study with respect to regulation of trs. DNA sequence analysis revealed identical -35 sequences in the predicted promoter regions for trsA, trsL, and trsN (Fig. 1 and Table 2). In addition, promoter sequences for trsA and trsL were contained within identical regions of dyad symmetry (Fig. 3). An abundant trsN transcript was seen by Northern blot analysis (Fig. 6), and a protein of the size predicted for TrsN was seen in the E. coli transcription-translation assay when only the trsN PCR product was used (40). We have prelim- inary evidence that the trsN gene product is at least one of the trs regulatory molecules and that one of its targets is the sequence immediately 5' to trsL (40). 
The organization and units of transcription of genes in- volved in conjugative transfer vary among other plasmid systems. On F and related plasmids from gram-negative organisms, transfer genes are clustered as a contiguous unit (49), as are the trs genes on pGOL. Even though the F system was the first and one of the best described, the organization of most of its transfer genes into a single major transcription unit appears to be uncommon. Broad-host-range IncP plas- mids have two separate transfer regions, one that contains genes for surface exclusion and formation of mating aggregates (Tra2) and another that encodes mostly genes involv- ing DNA transfer and metabolism (Tral). Furthermore, the genes in Tral seem to be arranged in at least four individual transcription units (47) while a single primary promoter transcribes the Tra2 genes (23). Other conjugative plasmids from gram-negative organisms, such as those of the IncN, IncP10, IncH, and Incla incompatibility groups, as well as Agrobacterium Ti plasmids (16, 27, 36, 42, 51), also have conjugative transfer functions segregated into two or more distinct regions that differ in transcriptional organization. Among other conjugative plasmids from gram-positive or- ganisms, there also appear to be separate transfer regions. Enterococcal pheromone-responsive plasmids have individ- ual regions that contain genes for DNA transfer and mating aggregation (pheromone response), and broad-host-range streptococcal plasmid pIPS01 has two separate clusters of transfer genes with undefined functions (20, 48). Thus, trs seems to be unlike the most prevalent broad-host-range plasmids from gram-negative organisms and more similar to F in that 8 of the 14 transfer genes are within a single transcription unit and all transfer genes are clustered. How- ever, since there are no surface appendages (26) and no aggregation substances (5) on staphylococcal cells that con- tain pGO1, most of the trs genes may be involved in DNA transfer rather than in mating aggregation. Therefore, trs may be more similar to the 10-to 15-kb gene clusters on broad-host-range plasmids from gram-negative species and pheromone-responsive plasmids from gram-positive species that function primarily in conjugal DNA metabolism and transfer. 
Some additional evidence for the similarity of trs and some of the transfer genes from gram-negative organisms came from the comparison of predicted trs gene products with those in the data base. The amino acid similarity among the predicted products of the transfer genes of gram-negative species, including virD4, traD, and traG, has recently been noted by Lessl and colleagues (24). The trsK gene product is also related to the predicted products of these genes of gram-negative organisms and has a consensus sequence for a type B NTP-binding site in common with these genes. The traD gene product has been localized to the cell membrane of E. coli and has been proposed either to guide the DNA through the cell membrane or to link the relaxasome with other membrane proteins that serve to form a membrane pore (23). traG and virD4 are grouped in operons that contain genes involved in DNA relaxation (47), suggesting that traD, traG, and virD4 serve to link DNA transfer with single-strand nicking. However, no other trs genes have significant similarity to gram-negative conjugative transfer genes in the data base and there is no formal evidence that pGO1 moves from donor to recipient cells as a single strand. The only indirect evidence for single-strand transfer comes from experiments showing that pGO1 mobilization of small, nonconjugative plasmid pC221 from staphylococci requires pC221 relaxation and that relaxation involves two pC221encoded genes, mobA and mobB (32). mobA encodes DNA- nicking activity, while mobB is, presumably, a single-strand binding protein. It is of interest, therefore, that the only other trs gene with a predicted product showing amino acid similarity to a transfer gene product was trsC, with homol- ogy to the pC221 mobB product. This further strengthens the theory that single-strand transfer is the operative mechanism for pGOL. 
While a number of studies have assessed conjugation systems in gram-negative bacteria, similar studies are infre- quent for conjugative plasmids in gram-positive cocci. Given the growing importance of staphylococci as nosocomial pathogens, largely because of their acquisition of antimicro- bial resistance genes, it seems important to characterize gene transfer among these organisms. This study provides the first complete DNA sequence and transcriptional analy- sis of gram-positive conjugative transfer genes and gives some indication that genetic organization differs in many ways from the organization reported for other transfer systems. It should serve as a basis for further studies of gene function and regulation. 

Remarks and source or reference Recombination-defective host; 8 9 S

 89 Allows blue-white color selection for pBluescriptlIKS(+) plasmids; 




  Q EI SN LT K KK KAK A LL 243 GSIR KITI I YVINILPIIA GNTVI GAAATTTAACGAVCAAGATATAACTCATTTAACTAAAAAAAA AASCACATTATTACT 11201 GGATTAAGAAAAACTATTATAATATG0TAATTACCAGTTTTGAGGAAACACKGAI I N E K T A I W N K E I Y K T I V K K P A 277 N I A S K N A A N I P V P V S 16 CR K YTAI SIT T i all 




  V PNRAP0NSN T 13201 TITATITITCTTTAACAIAACTATACCTAGWAGTTTIIAIATAAT"AAAACD 59 D Y A S K A N I I I I K A 5 1 E T K K N I S N V V NV I K K 13601 TGAAGAAAAA6AGAAAAACTTAATTGVAAT60TAGATAAATACACTAGAAGA6 43 A I V N N N N I v I q s * 




  N I N N 8 R C L AAAASGCATATNATT NCTAAGT iCTATAAAACCA6MSAI-AT --C A I 8 P A E D S C 11 D It S I S D ------'-'--''-'-'-------------'--'---'*- 


ACAAAAGGA&TTATATTACTAMTGACATGTCATGTGTAAATGTGGTATTGTGTTThAa

  FIG. 4.Autoradiogram of E. coli S30 coupled transcription- translation studies. Lanes: A to D, labelled proteins produced by specific DNA used for the reaction; A, pBR322; B, pGO203C; C, pGO200; D, pGO210. Rainbow molecular weight markers (Amersham) were used to size proteins, and the migration of these bands is indicated by dashes and the corresponding molecular weights (MW). Arrows with letters indicate the trs-encoded proteins corre- sponding to the protein bands visualized. Arrows with asterisks indicate the 13-lactamase protein (31.5 kDa) and its precursor form (114 kDa). The band above trsE (e) in lanes B and C did not correspond to any of the predicted trs-encoded proteins but may be a vector-encoded protein that is visualized faintly in lane A. A trsD-encoded protein (d in lanes B and C) was revealed by Western immunoblotting of the proteins produced in lanes B and C by using polyclonal antisera generated against the purified trsD gene product. 


FIG. 5 .

 5 FIG.5. Results of complementation studies. (A) Schematic representation of pGO200 containing Tn917 or Tn9171ac insertions. The trs ORF that corresponds to each insert is diagrammed above pGO200, and the subclones used for these experiments are diagrammed below pGO200. Only ORFs containing transposon inserts are included. All ORFs are shown in Fig.2. Restriction enzyme abbreviations are as described in the legend to Fig.2. (B) Subclones used for complementation are in the left-hand column. Tn917 and Tn91l7ac insertion numbers and the trs ORF into which each inserted (in parentheses) are listed at the top of each column. +, >200 transconjugates per mating; -, 0 to 10 transconjugates per mating; ND, not determined since the corresponding region of DNA is not contained within the subclone. 


5 FIG. 6 .

 56 FIG. 6. Autoradiogram of Northern blot analysis. (A to G) Total cellular mRNA preparations of S. aureus pGOl/4220 separated through 1.2% agarose containing 2.2 M formaldehyde. DNA probes and relative exposure times: lane A, trsN PCR product, 23 h; B, pGO187 DNA, 23 h; C, trsB PCR product, 23 h; D, trsL PCR product, 23 h; E, trsM PCR product, 23 h; F, pGO137 DNA (gentamicin resistance gene probe [aac-aph]), 7 h; G, protein A gene probe DNA, 3 h. The specific radioactivity of each probe and the amount of RNA were the same for each lane. -, rRNA bands.Arrows, easily identified transcript. 1, 400-base trsN transcript; 2, 1,300-base trsA-trsB transcript; 3, 1,800-base trsA-trsC transcript; 4, 1,500-base trsL-trsM transcript; 5, 1,800-base gentamicin resistance gene transcript; 6, 1,500-base protein A gene transcript. Sizes of transcripts were determined by comparisons to RNA molecular weight standards (not shown). Northern blot procedures were repeated several times, and identical results were obtained each time. The band hybridizing below transcript 2 in lane C was not explainable by sequence or complementation data but was consistently seen. 


TABLE 1 .

 1 Bacterial strains and plasmidsChromosomal genotype or phenotypea 





  J.BACTERIOL.from https://journals.asm.org/journal/jb on 24 September 2023 by 149.165.155.222. 


	62 101 caacagaccATCTTTACTGCTTTTGAGTTAT Downloaded gatctaCggattttCgccatgccacgattaC 50

TABLE 2 .

 2 Summary of ts ORFs 


	ORF	Size (bp)	Protein (Da)a	SD sequence and translational startP Promoter sequences (5'3')
	A B	975 315	37,463 11,318	CAATAGAAAAGT5G-ATTGTAA ATATAAGAAGTTGATTAATTAIN	-35 TGTCATIACAAAAAAGTTAAAAAAGTTTTATMTTTA (P2)C (-35) -10 TACTATG GA(Pl)C
	C	408	15,476	GTTTTAAAAATGGATGAATTATAI	
	D E F G H I J K	684 2,019 1,281 1,077 486 2,103 465 1,641	26,766 77,838 50,187 38,387 18,763 80,757 17,437 62,672	AAAAGTAAAMATTGGGTGAI AGA CA A-G TTT ATG TTTATGAATAQA9TAATCTCAI AATAGGAG GA9IAAAGTATCAJ TTATGATAAGTAGGTA A M AGATAGAAAMOMTAATACTTAf CGATTAAAAGGAUATAAGAAAGTG TAGAAGAAADGA&GATAAGGAAT	-35 ATTTAGCAAAGGCAGAAAGTTTACTTCTATAAAAGT -10
	L M N	918 393 189	33,908 14,861 6,993	TATTGAAAGTAUGGTGGTTGA&M AAATAAAGAAAGGAQATATTTA	-35 ACATGACATTGTAATGCAAAATCTGTlACTT (-35) -10 -35 -10

Downloaded from https://journals.asm.org/journal/jb on 24 September 2023 by 149.165.155.222. 

L Downloaded from https://journals.asm.org/journal/jb on 24 September 2023 by 149.165.155.222. 

* I -P.-O.-Downloaded from https://journals.asm.org/journal/jb on 24 September 2023 by 149.165.155.222. 



* Downloaded from https://journals.asm.org/journal/jb on 

September 2023 by 149.165.155.222. 

J. BACT1ERIOL. Downloaded from https://journals.asm.org/journal/jb on 24 September 2023 by 149.165.155.222. 

ACKNOWLEDGMENTS

This study was supported by U.S. Public Health Service grant AI/GM21772 from the National Institute of Allergy and Infectious Diseases.
Teresa M Morton, Denise M Eaton,', J Linda Johnston, Gordon L Archer'
DNA Sequence and Units of Transcription of the Conjugative Transfer Gene Complex (trs) of Staphylococcus aureus Plasmid pGO1