Alterations in the carboxy-terminal half of cloacin destabilize the protein and prevent its export by Escherichia coli.

Several overlapping carboxy-terminal and internal deletions were constructed in the cloacin structural gene. The expression, the binding of the cloacin DF13 immunity protein and the release into the culture medium of the mutant cloacin polypeptides were studied by immunoblotting and ELISAs. Minor alterations at the carboxy-terminal end of the cloacin did not affect protein expression, stability or release to a large extent, but larger carboxy-terminal deletions strongly destabilized the protein and no release was observed. The removal of a particular region within the carboxy-terminal portion of cloacin strongly destabilized the polypeptide and made it a target for proteolytic degradation. Binding of immunity protein did not affect stability and release of the mutant polypeptides. By using immunoelectron microscopy, the polypeptides that were not exported were located in the cytoplasm of producing cells. Large aggregates of these mutant polypeptides were not observed in the cytoplasm: the polypeptides were present in a soluble form.

Uncoupling of synthesis and release of cloacin DF13 and its immunity protein by Escherichia coli.

The synthesis of the bacteriocin cloacin DF13 and its release into the culture medium were genetically uncoupled by subcloning the gene encoding the bacteriocin release protein (BRP) from pCloDF13. The gene was cloned under the control of the IPTG-inducible lpp-lac promoter-operator system on the expression vector pINIIIA1, giving pJL1. A 4 kb DNA fragment of pJL1, containing the tandem lpp-lac promoter, the BRP gene and lacI (BRP cassette), was cloned into the pCloDF13 derivative plasmid pJN67, which encodes cloacin DF13 but not the release protein. Furthermore, the pCloDF13 immunity protein gene was subcloned downstream of the temperature-inducible PL promoter of the expression vector pPLc236, together with the BRP cassette. Growth, induction and excretion experiments with Escherichia coli cells harbouring the constructed plasmids revealed that: the BRP is the only pCloDF13-derived gene product responsible for the observed growth inhibition and apparent lysis of strongly induced cells. This growth inhibition and lysis can be prevented by Mg2+ ions added to the culture medium, and involves induction of phospholipase A activity. The expression of the BRP gene can be regulated by varying the IPTG concentration. A separately controlled and moderate induced BRP synthesis can be used to bring about the release of large amounts of cloacin DF13 under conditions that allow a strong induction of the bacteriocin and which do not result in lysis of cells. Preliminary results indicated that the BRP can stimulate the release of immunity protein in the absence of cloacin or cloacin fragments.

Escherichia coli ribosomal protein S1 does not protect the 49-nucleotide 3' terminal cloacin fragment of 16 S rRNA from nuclease S1.

Footprinting of ribosomal protein S1 on the 49-nucleotide 3' terminal cloacin DF13 fragment of 16 S rRNA at physiological ionic strength, pH and temperature yielded no detectable protection of any nucleotides from subsequent attack by the single strand specific nuclease S1, even at large excesses of ribosomal protein S1.

Methylation-dependent transcription controls plasmid replication of the CloDF13 cop-1(Ts) mutant.

The CloDF13 cop-1(Ts) mutant expresses a temperature-dependent plasmid copy number. At 42 degrees C the mutant shows a "runaway" behavior, and cells harboring this plasmid are killed. The cop-1(Ts) mutation is a G-to-A transition that disturbs one of the two methylation sites which are located opposite in the stem-loop structure within a region involved in both the initiation of primer synthesis for DNA replication and the termination of the cloacin operon transcript. We demonstrate that the mutation results in an increased primer (RNA II) synthesis resulting from nonconditional enhanced RNA II promoter activity, which at 42 degrees C causes a decrease in the amount of active replication repressor molecules (RNA I) synthesized from the opposite strand. We found that the absence of Dam methylation abolishes the mutant phenotype and that under this condition the high mutant level of RNA II synthesis is reduced, which is accompanied by a restoration of the regulation by RNA I. The role of methylation in the regulation of plasmid replication is discussed.

Circular dichroism and 500-MHz proton magnetic resonance studies of the interaction of Escherichia coli translational initiation factor 3 protein with the 16S ribosomal RNA 3' cloacin fragment.

The RNA helix destabilizing properties of Escherichia coli initiation factor 3 protein (IF3), and its affinity for an evolutionarily conserved sequence at the 3' end of 16S rRNA, led us to examine the details of the protein-nucleic acid interactions upon IF3 binding to the 49-nucleotide 3'-terminal cloacin DF13 fragment of 16S rRNA by studying the circular dichroism (CD) and proton magnetic resonance spectra of the RNA, the protein, and their complex. In a physiological tris(hydroxymethyl)aminomethane buffer, where the interaction is primarily nonionic and sequence specific, addition of IF3 decreases the RNA 268-nm CD peak hyperbolically by 19% to an end point of about one IF3 per RNA strand. The titration curve is best fit by an association constant of (1.80 +/- 0.05) X 10(7) M-1, within the range estimated by a nuclease mapping study of the same system [Wickstrom, E. (1983) Nucleic Acids Res. 11, 2035-2052]. In a low-salt phosphate buffer without Mg2+, where the interaction is primarily ionic and nonspecific, titration with IF3 decreases the peak CD sigmoidally by 35% to an end point of two IF3 per strand. The titration curve is best fit by an intrinsic association constant of (1.7 +/- 0.7) X 10(6) M-1 for each IF3 and a cooperativity constant of 33 +/- 6. In a physiological phosphate buffer lacking Mg2+, the dispersion of aromatic proton magnetic resonance peaks and upfield-shifted methyl proton resonances indicates a high degree of secondary and tertiary structure in the protein. In an equimolar mixture of IF3 and RNA cloacin fragment, several changes in identifiable IF3 and RNA resonances are observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Excretion of proteins by gram-negative bacteria: export of bacteriocins and fimbrial proteins by Escherichia coli.

In gram-negative bacteria only few proteins are exported across both the cytoplasmic membrane and the outer membrane which forms an extra barrier for protein excretion. In this review we describe the mechanisms of production and export of two types of plasmid-encoded proteins in Escherichia coli. These proteins are the bacteriocin cloacin DF13 and the K88ab and K99 fimbrial subunits. Specific so-called helper proteins located at different positions in the cell envelope play an essential role in the export of these proteins. The genetic organization, subcellular location and functions of these helper proteins, as well as the effects of mutations and culture conditions on the export of the proteins are described. Models for the export mechanisms are presented and future application possibilities for engineering foreign protein excretion in E. coli with these export systems are discussed.

Nuclease mapping of the secondary structure of the 49-nucleotide 3' terminal cloacin fragment of Escherichia coli 16s RNA and its interactions with initiation factor 3.

Escherichia coli translational initiation factor 3 (IF3) may be crosslinked to the 3' end of 16S RNA in 30S ribosomal subunits. In order to determine the sequence to which IF3 may bind in vivo, samples of 5'-32P labelled 3' terminal 49-nucleotide fragment of 16S RNA were incubated 5 min. at 37 degrees in 40 mM Tris-HOAc, pH 7.4, 100 mM NaCl, 1 mM Mg (OAc)2, 1 mM ZnSO4, with or without IF3, then reacted a further 5 min with nuclease S1, RNase T1, or RNase A. Base pairing between the 5' and 3' legs of the fragment occurs in the absence of IF3, but is disrupted by IF3 binding. IF3 appears to protect some residues near the 5' end of the fragment (U1495, A1499, A1500, A1502, and A1503) from nuclease S1, and potentiates S1 attack on others (G1494, G1497, C1501, G1504, G1505, U1506, G1517, G1529, G1530, and C1533). A series of equimolar reactions at increasing dilution imply an association constant range of 1.4-7.0 X 10(7) M-1.

Protein H encoded by plasmid CloDF13 is involved in excretion of cloacin DF13.

Excretion of cloacin DF13 was studied in Escherichia coli cells harboring different CloDF13 insertion and deletion mutant plasmids. Insertions of a transposon at position 9.8 or 11.5% of the CloDF13 plasmid blocked the expression of gene H and strongly reduced the specific excretion of cloacin DF13 into the culture medium, but had no effect on the production of cloacin DF13. Insertions in or deletions of regions of the CloDF13 DNA upstream the cloacin operon did not affect the excretion or production of the bacteriocin. Introduction of a CloDF13 plasmid that encodes for the gene H product in cells harboring a CloDF13 plasmid with an insertion in gene H stimulated the excretion of cloacin DF13 significantly in mitomycin C-induced and in noninduced cultures. Cloacin DF13 in cloacinogenic cells that did not produce the gene H protein was found to be about 90% located in the cytoplasm. In cells that did produce the gene H product, about 30% of the cloacin DF13 molecules were found in the cytoplasm, about 18% were found in the periplasm, about 2% were in the membranes, and about 50% were located in the culture supernatant. Cyclic AMP stimulated the production but not the excretion of cloacin DF13 in cells cultivated in the presence of glucose.

The nucleotide sequence of the bacteriocin promoters of plasmids Clo DF13 and Co1 E1: role of lexA repressor and cAMP in the regulation of promoter activity.

Treatment of cells, harbouring the bacteriocinogenic plasmic Clo DF13 with mitomycin-C, which induces the cellular SOS response, results in a significantly increased transcription of the operon encoding the bacteriocin cloacin DF13, the immunity protein and the lysis protein H. The nucleotide sequences of the promoter regions and N-terminal parts of the bacteriocin genes of Clo DF13, Col E1 and the pMB1 derivative pBR324 have been determined. A comparison of these sequences with those of corresponding regions of the lexA, recA and uvrB genes revealed that the promoter regions of the bacteriocin genes studied contain binding sites for the lexA protein, which is the repressor of the E. coli DNA-repair system. Using both, a thermosensitive lexA host strain and a host with pACYC184 into which the lexA gene had been cloned, we were able to demonstrate, that in vivo the lexA protein is involved in the regulation of bacteriocin synthesis. From the data presented, we conclude that bacteriocin synthesis is controlled at least by the lexA repressor. It has been reported that also catabolite repression might play an essential role in the control of bacteriocin synthesis. Computer analysis of the DNA sequence data indicated that the promoter regions of both, the cloacin DF13 and colicin E1 genes contain potential binding sites for the cyclic AMP-cyclic AMP Receptor Protein complex.

Transcription of bacteriocinogenic plasmid CloDF13 in vivo and in vitro: structure of the cloacin immunity operon.

Escherichia coli minicells harboring plasmid CloDF13 synthesized at least 25 messenger ribonucleic acid (RNA) species; three of these RNAs, a 2,400-, a 2,200-, and a 100-nucleotide RNA, were synthesized in relatively large amounts. Using insertion and deletion mutants of CloDF13 as well as an RNA blotting technique, we could demonstrate that these three RNAs are transcripts from the CloDF13 DNA region from 0 to 40%. This region contains the cloacin and immunity genes and the genetic information involved in plasmid DNA replication. A transcription map of this region is presented and discussed. The data indicate that the cloacin and immunity genes were coordinately transcribed into messenger RNAs of about 2,400 and 2,200 nucleotides, which differ in length at their 3' terminus. RNA polymerase binding studies and in vitro transcription assays indicated that transcription of these genes initiates at a promoter located around 32% on the CloDF13 map. Furthermore, it is shown that a 100-nucleotide RNA is encoded by the CloDF13 DNA region between 7.7 and 8.8% on the plasmid genome; the synthesis of this RNA proceeds in a direction opposite to the transcription of the cloacin and immunity genes.

Changes in protein synthesis on mitomycin C induction of wild-type and mutant CloDF13 plasmids.

Mitomycin C treatment of Escherichia coli K-12 cells containing the nonconjugative plasmid CloDF13 resulted in inhibition of host chromosome protein synthesis and a high rate of synthesis of two CloDF13-specified proteins whose molecular weights correspond to cloacin and immunity protein. Five molecules of immunity protein were synthesized for each cloacin DF13 molecule. Mitomycin C-treated cells containing a copy mutant of CloDF13 made three to four times as much of each protein as cells containing wild-type CloDF13. CloDF13 plasmids that contained the transposon Tn1 were isolated. Two did not induce after mitomycin C treatment, failing both to inhibit host cell synthesis and to produce the two new proteins. In minicells, they showed reduced CloDF13-specified protein synthesis and produced three Tn1-specified proteins.