INTRODUCTION

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Facultatively pathogenic microorganisms differ largely with respect to the mechanisms by which they affect their host. However, they all face the common problem that they have to strictly distinguish between two totally different "lifestyles," i.e., between living as commensals or even free in nature and being involved in the process of colonizing tissues or blood, which demands an alternate equipment of cell surface components, exoproteins, and metabolic enzymes.

The gram-positive bacterium Enterococcus faecalis is a commensal of the intestine, but under different circumstances may infect the urinary tract, blood, or endocardium. The conditions indispensable for the infectious pathway are still unknown, and there is no common factor identified for all clinical isolates. However, sex pheromone plasmid-encoded aggregation substance is a widespread adhesin shown to be involved in the colonization of various tissues (21, 28, 30). In addition, it plays an essential role in the conjugative transfer of the sex pheromone plasmid on which it is encoded in that it confers a tight contact between donor and recipient cells, visible as large clumps. (For reviews of the sex pheromone system, see references 7, 10, and 37.)

Therefore, regulation of aggregation substance is on three different levels. Under normal growth conditions, its expression is totally shut down. During the operation of the infectious pathway, there may be various environmental factors inducing aggregation substance, among them a component of blood serum (23) and several antibiotics (16, 39). For conjugative plasmid transfer, the corresponding gene is transcribed in response to a plasmid-specific oligopeptide, called sex pheromone, secreted by recipient cells not containing the corresponding plasmid. The latter phenomenon has been known for a long time and has been investigated by several groups (7, 10, 37). Especially for two different plasmids, pAD1and pCF10, rather detailed data on the regulatory circuits are available. Surprisingly, despite a similar overall organization of the regulatory genes and highly homologous DNA regions (15), induction of aggregation substance seems to involve two mechanistically distinct strategies. While for the pAD1-encoded aggregation substance, Asa1, a trans-acting protein, TraE1, obviously serves as the general inducer of transcription (25, 32) (a survey of the genetic data available for pAD1 is given in Fig. 1), the pCF10-encoded aggregation substance, Asc10, is expressed via transcriptional readthrough which involves a cis-acting, orientation-dependent factor (6), a regulatory RNA molecule interacting with ribosomal proteins, and a small proteinaceous regulator (4, 5).

The question is whether two related plasmids may have developed totally different strategies to regulate the same adhesin, or whether there are common basic pathways only slightly modified to ensure a specific response to the corresponding sex pheromone. This study presents data on the regulation of sex pheromone plasmid pAD1, supporting in part the second idea and addressing the function of several pAD1-specific genes. These data were obtained by use of a newly constructed -amylase-based promoter-probe vector.

	MATERIALS AND METHODS

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Strains, plasmids, and growth conditions. E. faecalis strains OG1X (19) and OG1X(pAD1) were grown in Todd-Hewitt-broth (THB; Oxoid). Escherichia coli cloning strain TOP10F' (Invitrogen) was grown in Luria-Bertani broth (24). Selective antibiotics were added as follows: erythromycin, 800 µg/ml for E. coli and 20 µg/ml for E. faecalis; chloramphenicol, 20 µg/ml for both organisms.

Construction of pAMY-em1 vector and derivatives containing pAD1 fragments. pAMY-em1 is composed of the same genetic elements used for the construction of expression vector pERM-ex1 (27). In addition, a promoterless chloramphenicol acetyltransferase gene (cat) protected by a downstream terminator (1) was integrated adjacent to the polylinker, but in the opposite orientation relative to amyL.

Mutational inactivation of pAD1 genes. pAD1-encoded orf1 was mutated according to Kunkel et al. (22); with a single nucleotide exchange, a BamHI site was introduced that converted the fourth orf1 codon into a stop codon.

-Amylase activity assay. Overnight cultures of E. faecalis strains containing pAMY-em1 constructs were inoculated 1:100 into 1 ml of fresh THB medium supplemented with 20-µg/ml concentrations of chloramphenicol and erythromycin (with or without synthetic sex pheromone cAD1). After ca. 5 h of incubation at 37°C, cultures were placed on ice, and 200 µl of each cell suspension was removed for the determination of turbidity at a wavelength of 600 nm. (Note that to dissolve cell aggregates grossly influencing the turbidity, suspensions are mixed with 1 ml of 8 M urea; this treatment does not detectably lyse E. faecalis cells or break cell chains.) Turbidity should not exceed a value of about 0.7 to ensure that cells are still exponentially growing. The rest of the cultures were centrifuged at 4°C, and 700 µl of supernatant was mixed with 200 µl of a precooled slurry of Phadebas reagent (Pharmacia) in H₂O. The mixtures were heavily stirred at 75°C on an Eppendorf shaker (Thermomixer 5436) until not more than half of the slurry had lost its blue color (in order to guarantee linearity of activity values). The reaction was stopped by placing the suspensions on ice for several minutes, spinning them briefly in a cooled centrifuge, and transferring 700 µl of supernatant to a plastic cuvette containing 500 µl of 0.5 M NaOH (which stops the reaction). Optical densities at 620 nm (OD₆₂₀) were determined, and the resulting values were equalized to standard conditions (i.e., cell densities of OD₆₀₀ = 1.0 and 15 min of amylase reaction time).

Clumping assay. Supernatants from overnight cultures of OG1X strains containing pAMY-em1 constructs were boiled for 5 min to kill residual E. faecalis cells and diluted with fresh THB in steps of 1:2 with 24-well microtiter plates used as a reservoir. After 1:100 inoculation with an overnight culture of OG1X(pAD1/pRBS_amy) (the second plasmid required because supernatants contain chloramphenicol and erythromycin), the microtiter plates were gently shaken at 37°C for several hours, and the formation of macroscopically visible aggregates was determined.

Nucleotide sequence accession number. The nucleotide sequence of pAMY-em1 has been submitted to GenBank (accession no. AJ243541).

	RESULTS

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Construction of a new -amylase-based promoter probe vector. Previous analyses of asa1 regulation mainly relied on three different methods: (i) Northern blotting (2, 13, 25) (Fig. 1), (ii) clumping assays using aggregation substance itself as a marker (11, 25), and (iii) transcriptional lacZ fusions based on a Tn917-derivative bearing a 'lacZ gene at one end (29, 34). Transcriptional analysis is too time-consuming for routine investigations and difficult to quantify. Clumping assays are easy to execute, but quantification is even less sensitive than Northern blotting. The use of Tn917lac suffers from the random location of transposition sites and from the polar effect of the 8.5-kb insert inactivating the possibly essential downstream sequence. Furthermore, only the transposition site itself was analyzed for transcription, while the effects of mutations on distant DNA regions are difficult to address. Our own previous attempts to present the lacZ gene on a definite promoter probe vector failed because of the instability of constructs. In addition, for assaying very weak promoter activities, -galactosidase may be inadequate, since this cytoplasmic enzyme possibly is not quantitatively extracted from the highly rigid E. faecalis cells.

View larger version (22K): [in this window] [in a new window]   FIG. 1.   Genetic organization of a pheromone-regulated pAD1 region and functions of several gene products according to previous publications. Interrupted arrows indicate constitutive transcripts. m0 to m5, pheromone-inducible transcripts; Px, presumptive promoters as localized by primer extension experiments. rho-independent transcriptional terminators are indicated as stem-loop structures.

View larger version (23K): [in this window] [in a new window]   FIG. 2.   Promoter probe vector pAMY-em1 (GenBank accession no. AJ243541). amyL, promoterless -amylase gene from B. licheniformis; cat: promoterless chloramphenicol acetyltransferase gene from Staphylococcus aureus plasmid pC194; ermAM: erythromycin resistance gene from E. faecalis plasmid pAM1; T0, T1, TTS1, and TTS2, transcriptional terminators. Selected restriction sites present only once or twice are indicated, the latter in parentheses.

View larger version (36K): [in this window] [in a new window]   FIG. 3.   Survey of pAMY-em1 constructs used in this study. (Fusions involving the ermAM promoter [Perm] were constructed by use of the related vector pERM-ex1 [27].) The nomenclature of constructs is given as follows: genes are indicated by a one-letter code (B, traB; C, traC; A, traA; E, traE1; Y, orfY; and 1, orf1); promoters (Px) and terminator T1 (TTS1) are given only when they border disruptions of the original sequence; genes given in parentheses have been destroyed by point mutations (graphically marked by black triangles), while primes indicate the removal of a few nucleotides from the 5' or 3' ends, respectively. The diagrams of the constructs (in this and the following figures) are aligned to the graphical representation of the pAD1 sequence given on the top. Only complete genes (and those disrupted by point mutations) are accented by thick lines. Restriction sites involved in the various constructions are indicated.

traB and traC are both required for sex pheromone-dependent asa1 expression. In the first approach, the complete transcriptional units defined by the promoters determined previously for the regulatory region of pAD1 (13) were successively added to the asa1-amyL fusion (Fig. 4). P₃ and P_1/2 turned out to be largely inactive. With the addition of P₀-traE1, -amylase was constitutively expressed at a high level (pEYP₃1). These data are consistent with previous results showing that activation of the P₃ promoter (responsible for asa1 transcription) is dependent on the activity of traE1 which was transcribed from P₀ in the absence of the negative regulator TraA (25, 32). Addition of the traA gene (pAEYP₃1) reduced amyL expression only slightly; this is not surprising, since the strains all produce sex pheromone, cAD1, which inactivates TraA (12).

View larger version (25K): [in this window] [in a new window]   FIG. 4.   Effects of various pAD1 fragments confined by presumptive pAD1-specific promoters in the presence or absence of sex pheromone cAD1. Black bars, -amylase activities of noninduced E. faecalis OG1X cultures containing one of the pAMY-em1 constructs; grey bars, -amylase activities of E. faecalis cultures induced by synthetic pheromone cAD1 (with pheromone concentration about 100-fold of the minimal inductory concentration). Activities (units per liter) always refer to the constructs given immediately on the right.

asa1 regulation involves a cis-acting factor in an orientation-dependent manner. The most obvious discrepancy between the regulation models of pAD1 and pCF10 lies in the fact that pCF10 is submitted to the action of a cis-acting factor probably tracking along the DNA in an orientation-dependent manner (6), while for pAD1, the TraE1 protein was shown to act in trans as a diffusible activator of the P₃ promoter (25, 32). To verify the model for pAD1 by using the amyL-based assay system, a fragment of the constitutive pAEYP₃1 construct covering the complete sequence from traA to traE1 was inverted relative to the amyL gene (Fig. 5). A strain containing the resulting construct (pEA/YP₃1) failed to produce -amylase (irrespective of the presence of cAD1). Further removal of the P_1/2-orfY and P₃-orf1 regions (pEA/YP1, pEA/P₃1, and pEA/RBS_amy) did not restore -amylase activity. The possibility that the disruption of the original sequence necessary for the rearrangement may have affected an essential structure can be largely excluded, since the used NsiI site (i) lies within a sequence lacking any open reading frame (ORF) or striking nucleotide sequence motif and (ii) even in the highly active constructs contains some additional nucleotides from a polylinker used for intermediate cloning. Only when part of traA and its downstream terminator were deleted in addition (pEP₀/P₃1) could some basic -amylase activity be measured, probably representing the constitutive countertranscription from the bidirectional P₀ promoter responsible for permanent traA expression.

View larger version (22K): [in this window] [in a new window]   FIG. 5.   Nondisruptive inversion of the region from traA through traE1. The mode of inversion involving a HincII-NsiI fragment (or NheI-NsiI for EP0/P31) is indicated by broken arrows. (Since there is no difference between induced and noninduced cultures, only results for noninduced cultures are indicated in this and the subsequent figures.)

traE1, orfY, and orf1 regions are essential. There are at least three small ORFs carried on pAD1 which are not present on pCF10. While traE1 and orf1 are unique to pAD1 (and related cAD1-inducible plasmids) (15), orfY is ubiquitous for most sex pheromone plasmids, but at least in the special case of pCF10, it is disrupted by a stop codon, leaving only a truncated 198-bp ORF (prgT) (20). These ORFs might be responsible for pheromone specificity of induction. To test their involvement in asa1 regulation, each of them (in common with some bordering sequences) was independently deleted from the constitutive construct pAEYP₃1. As shown in Fig. 6a, none of them could be removed without a nearly complete loss of -amylase activity.

View larger version (25K): [in this window] [in a new window]   FIG. 6.   Deletions of traE1, orfY, and orf1 genes from the constitutive pAEYP31 construct. (a) Gross deletions covering a great part of the gene (traE1) or the complete DNA region (orfY or orf1). (b) Point mutations of traE1 and orf1 and 3' truncation of orfY (see Materials and Methods for details).

traE1, orfY, and orf1 gene products are essential. The essential roles of traE1, orfY, and orf1 regions need not necessarily be connected with the respective gene products, but might be dependent on other genetic information encoded on the corresponding sequence. It therefore was necessary to specifically mutate the peptides without grossly changing the nucleotide sequence. traE1 was mutated by the introduction of a 2-bp frameshift, and orf1 was mutated by creating a stop codon near its 5' end (see Materials and Methods). In the case of orfY, the complete 3' end downstream of the unique AciI site (84 bp) was removed. Again, OG1X strains containing any of these constructs [and a double mutant, pA(E)Y'P₃1, given in Fig. 3] largely failed to produce -amylase (Fig. 6b), which implies an indispensable positive involvement of all three translation products in asa1 expression.

orfY and traA contribute to negative regulation. Interestingly, orfY seems to exhibit additional negative effects: Fig. 7a shows a pair of constructs differing with respect to the presence of orfY. Here, expression was achieved by removal of transcriptional terminators TTS1 and TTS2 along with traE1. In these cases, the presence of orfY reduced rather than increased -amylase activity. The same may hold true for orf1, but measurable effects are so weak that activity values may be considered as nonsignificant (not shown). traE1 could not be tested for possible negative effects, since removal of one or both of the upstream terminators without deleting traE1 has toxic effects on E. faecalis.

View larger version (25K): [in this window] [in a new window]   FIG. 7.   Negative effects of orfY (a) and traA (b). Several pairs of constructs are compared, differing only with respect to the corresponding gene.

	DISCUSSION

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In the present study, it could be shown that regulation of pAD1-encoded aggregation substance at least involves a total of 10 kb upstream of the corresponding asa1 gene. All ORFs of >100 bp within this region, except for sea1 encoding the surface exclusion protein, are required for sex pheromone-controlled asa1 expression. On the other hand, several important structures were excluded from a detailed analysis: iad (8) coding for inhibitory sex pheromone; traD, which encodes immediately downstream from iad but is transcribed in the opposite direction (2, 33); and a small ORF immediately upstream of asa1, which encodes an RAPC amino acid motif conserved for most, if not all, sex pheromone plasmids (27). The roles of these ORFs were not addressed independently of the genes investigated in detail; therefore, their functions might further complicate the picture of pAD1 regulation presented here.

Mutation of any of the genes tested either results in constitutive expression (traB or traA) or totally shuts down expression (traC, traE1, orfY, or orf1). As for pCF10, regulation involves a cis-acting orientation-dependent factor encodedor at least initiating its functionwithin the 2.3-kb region covering traA through traE1, since its pure inversion results in a total loss of expression. In probably all cases, the proteins encoded by the ORFs rather than their nucleotide sequences are necessary for regulation. For orfY, a specific role of the deleted 3'-terminal 84 bp on the nucleotide level cannot totally be excluded, although there are no hints at all from the DNA sequence (e.g., repeats, conserved motifs, or possible secondary structures).

orfY (and possibly orf1) plays an ambiguous role in that it exhibits an additional negative effect. The function of TraA as a negative regulator of asa1 expression could be established; however, it was not sufficient for a complete repression, since it probably is inactivated by cAD1 still produced by the tested strainsprovided that the pheromone is internalized despite the lack of a specific uptake system (see below). If this is the case, nonspecific internalization must be rather efficient, since addition of cAD1 to the culture broth of OG1X(pAEYP₃1) does not significantly raise -amylase activity (Fig. 4).

The role of the remaining genes which have been investigated previously will have to be modified or newly defined according to the data presented. TraB efficiently represses aggregation substance, but not simply by avoiding self-induction via its assumed function as a repressor of cAD1 production. This can be excluded, since constructs containing traB still promote expression and secretion of active cAD1 in E. faecalis. Only the addition of traC, irrespective of the presence of traB, switches off cAD1 production, an effect that cannot simply be explained by the primary function of TraC lipoprotein as a pheromone-specific oligopeptide transporter. It may be argued that TraC surface protein traps all self-produced cAD1 from the supernatantbut why does this not induce asa1 expression (in the case of pBCAEYP₃1 [Fig. 4]) while externally added pheromone does?

Last but not least, and contrary to our own previous results (25), TraE1 cannot be simply a trans-acting protein directly effecting transcription from the P₃ promoter. If this were the case, the inversion of a fragment containing the complete traE1 gene, including its original upstream sequence, which covers the corresponding P₀ promoter, should not alter activity. Instead, this manipulation switches off asa1 expression, proving the involvement of an orientation-dependent mechanism, as has been shown for pCF10 (6).

The most puzzling result was that even complete pAD1 cannot activate the P₃ promoter in trans, a strategy which has proven successful previously (25, 26). The fact that pAD1 inactivates the otherwise constitutive pAMY-em1 constructs would imply the existence of a pAD1-encoded, trans-acting super-repressor. However, even the control plasmid (pP_ermRBS_amy) constitutively expressing amyL and not containing any pAD1 sequence is affected by pAD1, in that -amylase activity is reduced to about 50% of its normal value (data not shown). Maybe the cellular secretion system mediating -amylase export out of the cell is influenced by pAD1 (possibly via simple competition by pAD1-encoded surface proteins). This effect should be independent of pAD1-specific sequences and therefore cannot explain either why amyL preceded by pAD1 sequences is nearly completely switched off, contrary to the incomplete inactivation of pP_ermRBS_amy control plasmid.

Updating of the model for asa1 regulation. How can the data presented here and in previous publications be integrated into a conclusive regulation model? The following view combines all of the negative and positive effects shown for the various regulatory genes. Under noninducing conditions, transcription within the regulatory region is locked by a cooperative action of TraB and TraA. While TraA binds directly to the P₀ promoter region (32), TraB acts in a still unclear way (but not simply by the shutdown of sex pheromone production). OrfY and Orf1 proteins could occupy specific positions upstream from their own coding regions, thus contributing to repression by preventing accidental transcription events. Additional factors help to keep the TTS1 and TTS2 terminators locked. Inhibitory pheromone iAD1 (8) competes with trace amounts of the sex pheromone cAD1, possibly present in the environment or produced by the cell itself, and traD countertranscription (2) interferes with transcription from the P₀ promoter in the direction of traE1.