In Vivo Analysis of Sequence Requirements for Processing and Degradation of the Colicin A Lysis Protein Signal Peptide

The lipid modification and processing of a number of colicin lysis proteins take place exceedingly slowly and result in the release of a stable signal peptide. It is possible that this peptide or the presence of lipid-modified precursors which result from the slow processing plays a role in the release of colicins and in the quasilysis that occurs in induced colicinogenic cultures. We used in vitro mutagenesis and pulse-chase radiolabeling and immunoprecipitation to examine the reasons for the slow processing and signal peptide degradation reactions for the colicin A lysis protein (Cal). In one mutant, isoleucine 13 was replaced with serine, and in another, alanine 18, the last residue of the signal peptide, was replaced with glycine. In each case, the mutation caused a striking increase in the rate of maturation of the precursor, and in the case of the serine 13 derivative, the mutation also destabilized the signal peptide. A precursor containing both of these mutations was completely matured and its signal sequence degraded within seconds of its synthesis. The release of colicin A and the quasilysis of producing cultures were unchanged for each of these mutants, indicating that neither the stable signal peptide nor lipid-modified processing intermediates of Cal are required for either of these events in wild-type cells.     

Phospholipase-A-independent damage caused by the colicin A lysis protein during its assembly into the inner and outer membranes of Escherichia coli

The requirement for the activation of phospholipase A by the colicin A lysis protein (Cal) in the efficient release of colicin A by Escherichia coli cells containing colicin A plasmids was studied. In particular, we wished to determine if this activation is the primary effect of Cal or whether it reflects more generalized damage to the envelope caused by the presence of large quantities of this small acylated protein. E. coli tolQ cells, which were shown to be leaky for periplasmic proteins, were transduced to pldA and then transformed with the recombinant colicin A plasmid pKA. Both the pldA and pldA+ strains released large quantities of colicin A following induction, indicating that in these cells phospholipase A activation is not required for colicin release. This release was, however, still dependent on a functioning Cal protein. The assembly and processing of Cal in situ in the cell envelope was studied by combining pulse-chase labelling with isopycnic sucrose density gradient centrifugation of the cell membranes. Precursor Cal and lipid-modified precursor Cal were found in the inner membrane at early times of chase, and gave rise to mature Cal which accumulated in both the inner and outer membrane after further chase. The signal peptide was also visible on these gradients, and its distribution too was restricted to the inner membrane. Gradient centrifugation of envelopes of cells which were overproducing Cal resulted in very poor separation of the membranes. The results of these studies provide evidence that the colicin A lysis protein causes phospholipase A-independent alterations in the integrity of the E. coli envelope.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and functioning of the colicin E1 lysis protein: comparison with the colicin A lysis protein.

The colicin E1 lysis protein, CelA, was identified as a 3-kDa protein in induced cells of Escherichia coli K-12 carrying pColE1 by pulse-chase labeling with either [35S]cysteine or [3H]lysine. This 3-kDa protein was acylated, as shown by [2-3H]glycerol labeling, and seemed to correspond to the mature CelA protein. The rate of modification and processing of CelA was different from that observed for Cal, the colicin A lysis protein. In contrast to Cal, no intermediate form was detected for CelA, no signal peptide accumulated, and no modified precursor form was observed after globomycin treatment. Thus, the rate of synthesis would not be specific to lysis proteins. Solubilization in sodium dodecyl sulfate of the mature forms of both CelA and Cal varied similarly at the time of colicin release, indicating a change in lysis protein structure. This particular property would play a role in the mechanism of colicin export. The accumulation of the signal peptide seems to be a factor determining the toxicity of the lysis proteins since CelA provoked less cell damage than Cal. Quasi-lysis and killing due to CelA were higher in degP mutants than in wild-type cells. They were minimal in pldA mutants.     

Lipoprotein nature of the colicin A lysis protein: effect of amino acid substitutions at the site of modification and processing.

The colicin A lysis protein (Cal) is required for the release of colicin A to the medium by producing bacteria. This protein is produced in a precursor form that contains a cysteine at the cleavage site (-Leu-Ala-Ala-Cys). The precursor must be modified by the addition of lipid before it can be processed. The maturation is prevented by globomycin, an inhibitor of signal peptidase II. Using oligonucleotide-directed mutagenesis, the alanine and cystein residues in the -1 and +1 positions of the cleavage site were replaced by proline and threonine residues, respectively, in two different constructs. Both substitutions prevented the normal modification and cleavage of the protein. The marked activation of the outer membrane detergent-resistant phospholipase A observed with wild-type Cal was not observed with the Cal mutants. Both Cal mutants were also defective for the secretion of colicin A. In one mutant, the signal peptide appeared to be cleaved off by an alternative pathway involving signal peptidase I. Electron microscope studies with immunogold labeling of colicin A on cryosections of pldA and cal mutant cells indicated that the colicin remains in the cytoplasm and is not transferred to the periplasmic space. These results demonstrate that Cal must be modified and processed to activate the detergent-resistant phospholipase A and to promote release of colicin A.     

The acylated precursor form of the colicin A lysis protein is a natural substrate of the DegP protease.

The acylated precursor form of the colicin A lysis protein (pCalm) is specifically cleaved by the DegP protease into two acylated fragments of 6 and 4.5 kilodaltons (kDa). This cleavage was observed after globomycin treatment, which inhibits the processing of pCalm into mature colicin A lysis protein (Cal) and the signal peptide. The cleavage took place in lpp, pldA, and wild-type strans carrying plasmids which express the lysis protein following SOS induction and also in cells containing a plasmid which expresses it under the control of the tac promoter. Furthermore, the DegP protease was responsible for the production of two acylated Cal fragments of 3 and 2.5 kDa in cells carrying plasmids which overproduce the Cal protein, without treatment with globomycin. DegP could also cleave the acylated precursor form of a mutant Cal protein containing a substitution in he amino-terminal portion of the protein, but not that of a mutant Cal containing a frameshift mutation in its carboxyl-terminal end. The functions of Cal in causing protein release, quasi-lysis, and lethality were increased in degP41 cells, suggesting that mature Cal was produced in higher amounts in the mutant than in the wild type. These effects were limited in cells deficient in phospholipase A. Interactions between the DegP protease and phospholipase A were suggested by the characteristics of degP pldA double mutants.     

Molecular characterisation of the colicin E2 operon and identification of its products

Summary The DNA sequence of the entire colicin E2 operon was determined. The operon comprises the colicin activity gene, ceaB, the colicin immunity gene, ceiB, and the lysis gene, celB, which is essential for colicin release from producing cells. A potential LexA binding site is located immediately upstream from ceaB, and a rho-independent terminator structure is located immediately downstream from celB. A comparison of the predicted amino acid sequences of colicin E2 and cloacin DF13 revealed extensive stretches of homology. These colicins have different modes of action and recognise different cell surface receptors; the two major regions of heterology at the carboxy terminus, and in the carboxy-terminal end of the central region probably correspond to the catalytic and receptor-recognition domains, respectively. Sequence homologies between colicins E2, A and E1 were less striking, and the colicin E2 immunity protein was not found to share extensive homology with the colicin E3 or cloacin DF13 immunity proteins. The lysis proteins of the ColE2, ColE1 and CloDF13 plasmids are almost identical except in the aminoterminal regions, which themselves have overall similarity with lipoprotein signal peptides. Processing of the ColE2 prolysis protein to the mature form was prevented by globomycin, a specific inhibitor of the lipoprotein signal peptidase. The mature ColE2 lysis protein was located in the cell envelope. The results are discussed in terms of the functional organisation of the colicin operons and the colicin proteins, and the way in which colicins are released from producing cells.     

Proteaselike sequence in hepatitis B virus core antigen is not required for e antigen generation and may not be part of an aspartic acid-type protease.

The hepatitis B virus (HBV) C gene directs the synthesis of two major gene products: HBV core antigen (HBcAg[p21c]), which forms the nucleocapsid, and HBV e antigen (HBeAg [p17e]), a secreted antigen that is produced by several processing events during its maturation. These proteins contain an amino acid sequence similar to the active-site residues of aspartic acid and retroviral proteases. On the basis of this sequence similarity, which is highly conserved among mammalian hepadnaviruses, a model has been put forward according to which processing to HBeAg is due to self-cleavage of p21c involving the proteaselike sequence. Using site-directed mutagenesis in conjunction with transient expression of HBV proteins in the human hepatoma cell line HepG2, we tested this hypothesis. Our results with HBV mutants in which one or two of the conserved amino acids have been replaced by others suggest strongly that processing to HBeAg does not depend on the presence of an intact proteaselike sequence in the core protein. Attempts to detect an influence of this sequence on the processing of HBV P gene products into enzymatically active viral polymerase also gave no conclusive evidence for the existence of an HBV protease. Mutations replacing the putatively essential aspartic acid showed little effect on polymerase activity. Additional substitution of the likewise conserved threonine residue by alanine, in contrast, almost abolished the activity of the polymerase. We conclude that an HBV protease, if it exists, is functionally different from aspartic acid and retroviral proteases.     

Delineation of the translocation of colicin E7 across the inner membrane of <Emphasis Type="Italic">Escherichia coli</Emphasis>

The lysis protein of the colicinogenic operon is essential for colicin release and its main function is to activate the outer membrane phospholipase A (OMPLA) for the traverse of colicin across the cell envelope. However, little is known about the involvement of the lysis protein in the translocation of colicin across the inner membrane into the periplasm. The introduction of specific point mutations into the lipobox or sorting signal sequence of the lysE7 gene resulted in the production of various forms of lysis proteins. Our experimental results indicated that cells with wild-type mature LysE7 protein exhibited higher efficiency of colicin E7 translocation across the inner membrane into the periplasm than those with premature LysE7 protein. Moreover, the degree of permeability of the inner membrane induced by the mature LysE7 protein was significantly increased as compared to the unmodified LysE7 precursor. These results suggest that the efficiency of colicin movement into the periplasm is correlated with the increase in inner membrane permeability induced by the LysE7 protein. Thus, we propose that mature LysE7 protein has two critical roles: firstly mediating the translocation of colicin E7 across the inner membrane into the periplasm, and secondly activating the OMPLA to allow colicin release.     

DNA and amino acid sequence analysis of structural and immunity genes of colicins Ia and Ib.

The nucleotide sequences for colicin Ia and colicin Ib structural and immunity genes were determined. The two colicins each consist of 626 amino acid residues. Comparison of the two sequences along their lengths revealed that the two colicins are nearly identical in the N-terminal 426 amino acid residues. The C-terminal 220 amino acid residues of the colicins are only 60% identical, suggesting that this is the region most likely recognized by their cognate immunity proteins. The predicted proteins for the colicin immunity proteins would contain 111 amino acids for the colicin Ia immunity protein and 115 amino acids for the colicin Ib immunity protein. The colicin immunity proteins have no detectable DNA or amino acid homology but do exhibit a conservation of overall hydrophobicity. The colicin immunity genes lie distal to and in opposite orientation to the colicin structural genes. The colicin Ia immunity protein was purified to apparent homogeneity by a combination of isoelectric focusing and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of the purified Ia immunity protein was determined and was found to be in perfect agreement with that predicted from the DNA sequence of its structural gene. The Ia immunity protein is not a processed membrane protein.     

Amino acid substitutions in pilin of Pseudomonas aeruginosa. Effect on leader peptide cleavage, amino-terminal methylation, and pilus assembly.

A total of 37 separate mutants containing single and multiple amino acid substitutions in the leader and amino-terminal conserved region of the Type IV pilin from Pseudomonas aeruginosa were generated by oligonucleotide-directed mutagenesis. The effect of these substitutions on the secretion, processing, and assembly of the pilin monomers into mature pili was examined. The majority of substitutions in the highly conserved amino-terminal region of the pilin monomer had no effect on piliation. Likewise, substitution of several of the residues within the six amino acid leader sequence did not affect secretion and leader cleavage (processing), including replacement of one or both of the positively charged lysine residues with uncharged or negatively charged amino acids. One characteristic of the Type IV pili is the presence of an amino-terminal phenylalanine after leader peptide cleavage which is N-methylated prior to assembly of pilin monomers into pili. Substitution of the amino-terminal phenylalanine with a number of other amino acids, including polar, hydrophobic, and charged residues, did not affect proper leader cleavage and subsequent assembly into pili. Amino-terminal sequencing showed that the majority of substitute residues were also methylated. Substitution of the glycine residue at the -1 position to the cleavage site resulted in the inability to cleave the prepilin monomers and blocked the subsequent assembly of monomers into pili. These results indicate that despite the high degree of conservation in the amino-terminal sequences of the Type IV pili, N-methylphenylalanine at the +1 position relative to the leader peptide cleavage site is not strictly required for pilin assembly. N-Methylation of the amino acids substituted for phenylalanine was shown to have taken place in four of the five mutants tested, but it remains unclear as to whether pilin assembly is dependent on this modification. Recognition and proper cleavage of the prepilin by the leader peptidase appears to be dependent only on the glycine residue at the -1 position. Cell fractionation experiments demonstrated that pilin isolated from mutants deficient in prepilin processing and/or assembly was found in both inner and outer membrane fractions, indistinguishable from the results seen with the wild type.     

A functional core of the mitochondrial genome maintenance protein Mgm101p in Saccharomyces cerevisiae determined with a temperature-conditional allele

Analysis of Mgm101p isolated from mitochondria shows that the mature protein of 27.6 kDa lacks 22 amino acids from the N-terminus. This mitochondrial targeting sequence has been incorporated in the design of oligonucleotides used to determine a functional core of Mgm101p. Progressive deletions, although retaining the targeting sequence, reveal that 76 N-terminal and six C-terminal amino acids of Mgm101p can be removed without altering the ability to complement an mgm101-1(ts) temperature-sensitive mutant. However, this active core is unable to complement mgm101 null mutants, suggesting that the Mgm101p might need to form a dimer or multimer to be functional in vivo. The active core, enriched in basic residues, contains 165 amino acids with a pI of 9.2. Alignment with 22 Mgm101p sequences from other lower eukaryotes shows that a number of amino acids are highly conserved in this region. Random mutagenesis confirms that certain critical amino acids required for function are invariant across the 23 proteins. Searches in the PFAM database revealed a low level of structural similarity between the active core and the Rad52 protein family.     

Colicin U, a novel colicin produced by Shigella boydii.

A novel colicin, designated colicin U, was found in two Shigella boydii strains of serovars 1 and 8. Colicin U was active against bacterial strains of the genera Escherichia and Shigella. Plasmid pColU (7.3 kb) of the colicinogenic strain S. boydii M592 (serovar 8) was sequenced, and three colicin genes were identified. The colicin U activity gene, cua, encodes a protein of 619 amino acids (Mr, 66,289); the immunity gene, cui, encodes a protein of 174 amino acids (Mr, 20,688); and the lytic protein gene, cul, encodes a polypeptide of 45 amino acids (Mr, 4,672). Colicin U displays sequence similarities to various colicins. The N-terminal sequence of 130 amino acids has 54% identity to the N-terminal sequence of bacteriocin 28b produced by Serratia marcescens. Furthermore, the N-terminal 36 amino acids have striking sequence identity (83%) to colicin A. Although the C-terminal pore-forming sequence of colicin U shows the highest degree of identity (73%) to the pore-forming C-terminal sequence of colicin B, the immunity protein, which interacts with the same region, displays a higher degree of sequence similarity to the immunity protein of colicin A (45%) than to the immunity protein of colicin B (30.5%). Immunity specificity is probably conferred by a short sequence from residues 571 to residue 599 of colicin U; this sequence is not similar to that of colicin B. We showed that binding of colicin U to sensitive cells is mediated by the OmpA protein, the OmpF porin, and core lipopolysaccharide. Uptake of colicin U was dependent on the TolA, -B, -Q, and -R proteins. pColU is homologous to plasmid pSB41 (4.1 kb) except for the colicin genes on pColU. pSB41 and pColU coexist in S. boydii strains and can be cotransformed into Escherichia coli, and both plasmids are homologous to pColE1.     

Synthesis and sequence-specific proteolysis of a hybrid protein (colicin A::growth hormone releasing factor) produced in Escherichia coli

DNA constructs coding for human growth hormone (hGH)-releasing factor (hGRF) preceded by the specific recognition sequence for the activated blood coagulation factor X (FXa), fused in frame to the N-terminal 172-amino acid residues of colicin A, have been expressed in Escherichia coli. The construct was placed under the control of the inducible caa promoter in an operon containing a downstream gene coding for the cell lysis protein, Cal. Induction resulted in excretion of only the processed colicin A fragment. Replacement of Cal by the terminator from phage fd resulted in high expression of the hybrid protein, which was recovered as cytoplasmic aggregates. Enzymatic cleavage of the purified and renatured hybrid protein using FXa allowed the recovery of authentic hGRF.     

Nucleotide sequence of the colicin B activity gene cba: consensus pentapeptide among TonB-dependent colicins and receptors.

Colicin B formed by Escherichia coli kills sensitive bacteria by dissipating the membrane potential through channel formation. The nucleotide sequence of the structural gene (cba) which encodes colicin B and of the upstream region was determined. A polypeptide consisting of 511 amino acids was deduced from the open reading frame. The active colicin had a molecular weight of 54,742. The carboxy-terminal amino acid sequence showed striking homology to the corresponding channel-forming region of colicin A. Of 216 amino acids, 57% were identical and an additional 19% were homologous. In this part 66% of the nucleotides were identical in the colicin A and B genes. This region contained a sequence of 48 hydrophobic amino acids. Sequence homology to the other channel-forming colicins, E1 and I, was less pronounced. A homologous pentapeptide was detected in colicins B, M, and I whose uptake required TonB protein function. The same consensus sequence was found in all outer membrane proteins involved in the TonB-dependent uptake of iron siderophores and of vitamin B12. Upstream of cba a sequence comprising 294 nucleotides was identical to the sequence upstream of the structural gene of colicin E1, with the exception of 43 single-nucleotide replacements, additions, or deletions. Apparently, the region upstream of colicins B and E1 and the channel-forming sequences of colicins A and B have a common origin.     

Replacement by site-directed mutagenesis indicates a role for histidine 170 in the glutamine amide transfer function of anthranilate synthase

Anthranilate synthase is a glutamine amidotransferase that catalyzes the first reaction in tryptophan biosynthesis. Conserved amino acid residues likely to be essential for glutamine-dependent activity were identified by alignment of the glutamine amide transfer domains in four different enzymes: anthranilate synthase component II (AS II), p-aminobenzoate synthase component II, GMP synthetase, and carbamoyl-P synthetase. Conserved amino acids were mainly localized in three clusters. A single conserved histidine, AS II His-170, was replaced by tyrosine using site-directed mutagenesis. Glutamine-dependent enzyme activity was undetectable in the Tyr-170 mutant, whereas the NH3-dependent activity was unchanged. Affinity labeling of AS II active site Cys-84 by 6-diazo-5-oxonorleucine was used to distinguish whether His-170 has a role in formation or in breakdown of the covalent glutaminyl-Cys-84 intermediate. The data favor the interpretation that His-170 functions as a general base to promote glutaminylation of Cys-84. Reversion analysis was consistent with a proposed role of His-170 in catalysis as opposed to a structural function. These experiments demonstrate the application of combining sequence analyses to identify conserved, possibly functional amino acids, site-directed mutagenesis to replace candidate amino acids, and protein chemistry for analysis of mutationally altered proteins, a regimen that can provide new insights into enzyme function.     

Identification of a novel eosinophil chemotactic cytokine (ECF-L) as a chitinase family protein

A novel eosinophil chemotactic cytokine (ECF-L) was purified from the culture supernatant of splenocytes of mice by a combination of anion-exchange chromatography, Procion red-agarose affinity chromatography, size exclusion high performance liquid chromatography (HPLC), and reverse phase HPLC. The NH(2)-terminal amino acid sequence was determined by direct protein sequencing. An ECF-L cDNA clone of 1,506 nucleotides was isolated from a cDNA library, and the nucleotide sequence predicted a mature protein of 397 amino acids. A recombinant ECF-L showed a level of eosinophil chemotactic activity comparable with that of natural ECF-L, and the activity was inhibited by a monoclonal antibody to ECF-L. ECF-L also attracted T lymphocytes and bone marrow polymorphonuclear leukocytes in vitro, whereas it caused selective extravasation of eosinophils in vivo. ECF-L mRNA was highly expressed in spleen, bone marrow, lung, and heart. A comprehensive GenBank data base search revealed that ECF-L is a chitinase family protein. ECF-L retains those amino acids highly conserved among chitinase family proteins, but Asp and Glu residues essential for the proton donation in hydrolysis were replaced by Asn and Gln, respectively. Although ECF-L contains a consensus CXC sequence near the NH(2) terminus akin to chemokine family proteins, the rest of ECF-L shows poor homology with chemokines.