Analysis of the in vivo activation of hemolysin (HlyA) from Escherichia coli

Ludwig, Albrecht; Garcı́a, Fernando; Bauer, Susanne; Jarchau, Thomas; Benz, Roland; Hoppe, Jürgen; Goebel, Werner

doi:10.1128/jb.178.18.5422-5430.1996

Cited by 67 publications

(82 citation statements)

References 48 publications

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“…4, lanes d and c). Moreover, HlyC was detected in cell lysates from a strain encoding HlyC and another form of HlyA (HlyA ⌬722-724, Table I), lacking part of a putative HlyC binding domain (26,27) (Fig. 4, lane a).…”

Section: In Vivo Proteolytic Degradation Of the E Coli Hlycmentioning

confidence: 99%

“…XL1BluepFG1a and XL1BluepUC18 were induced with IPTG as described (26). Cell lysates from both strains were obtained by resuspension in 2ϫ SDS-PAGE sample buffer.…”

Section: Identification Of Proteins By Maldi-mass Spectrometry and Pementioning

confidence: 99%

“…26. Seventy g of total protein obtained after sonication of bacteria were mixed with 500 l of immunoprecipitation buffer (150 mM NaCl, 1% Triton X-100, 10 mM Tris, pH 7.4, 1 mM EDTA, and 0.2 mM NaO 3 V) as described (36).…”

Section: Construction Of Recombinant Dna Expression and Purification Ofmentioning

confidence: 99%

“…The pellet obtained after centrifugation was washed twice with ice-cold acetone and stored for no longer than 1 day at Ϫ20°C. For analysis on two-dimensional polyacrylamide gel electrophoresis, the protocol described by Ludwig et al (26) was followed with some modifications. Briefly, precipitated culture supernatants were resuspended in 20 l of isoelectrofocusing sample buffer (62.5 mM Tris-HCl, pH 6.8, 0.5% SDS, and 10% glycerol) and 2 l of 1 M Tris-HCl, pH 9.0.…”

Section: Construction Of Recombinant Dna Expression and Purification Ofmentioning

confidence: 99%

“…The unique acylation reaction catalyzed by HlyC does not involve covalent attachment of fatty acid molecules to the acyltransferase but a direct interaction between acylACP-HlyC and pro-HlyA through a non-covalent ternary complex (22). A HlyC binding domain in pro-HlyA has been proposed (26,27), and some highly conserved amino acid residues in the carboxyl and amino termini of HlyC essential for the acylation process have been identified (17, 28 -30).…”

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confidence: 99%

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In Vivo Proteolytic Degradation of the Escherichia coli Acyltransferase HlyC

Guzmán-Verri

Chaves-Olarte

Garcı́a

et al. 2001

Journal of Biological Chemistry

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Escherichia coli hemolysin (HlyA) is the prototype toxin of a major family of exoproteins produced by Gram-negative bacteria known as "repeats in toxins." Only fatty acid-acylated HlyA molecules at residues Lys 564 and Lys 690 are able to damage the target cell membrane. Fatty acylation of pro-HlyA is dependent on the co-synthesized acyltransferase HlyC and the acylated form of acyl-carrier protein. By using a collection of hlyA and hlyC mutant strains, the processing of HlyC was investigated. HlyC was not detected by Western blot in an E. coli strain encoding hlyC and hlyA, but it was present in a strain encoding only hlyC. The hlyC mRNA pattern, however, was similar in both strains indicating that the turnover of HlyC does not occur at the transcriptional level. HlyC was detected in Western blots of cell lysates from an E. coli strain encoding HlyC and a HlyA derivative where both acylation sites were substituted. Similar results were obtained when HlyC was expressed in a hlyA mutant strain lacking part of a putative HlyC binding domain, indicating that this particular HlyA region affects HlyC stability. We did not detect HlyC in cell lysates from hlyC mutants with different abilities to acylate pro-HlyA, suggesting that the degradation of HlyC is not related to the HlyA acylation process. The protease systems ClpAP, ClpXP, and FtsH were found to be responsible for the HlyA-dependent processing of HlyC.

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Section: In Vivo Proteolytic Degradation Of the E Coli Hlycmentioning

confidence: 99%

“…XL1BluepFG1a and XL1BluepUC18 were induced with IPTG as described (26). Cell lysates from both strains were obtained by resuspension in 2ϫ SDS-PAGE sample buffer.…”

Section: Identification Of Proteins By Maldi-mass Spectrometry and Pementioning

confidence: 99%

Section: Construction Of Recombinant Dna Expression and Purification Ofmentioning

confidence: 99%

Section: Construction Of Recombinant Dna Expression and Purification Ofmentioning

confidence: 99%

mentioning

confidence: 99%

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In Vivo Proteolytic Degradation of the Escherichia coli Acyltransferase HlyC

Guzmán-Verri

Chaves-Olarte

Garcı́a

et al. 2001

Journal of Biological Chemistry

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Mass spectrometric analysis of recombinant adenylate cyclase toxin from Bordetella pertussis strain 18323/pHSP9

et al. 2001

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The adenylate cyclase toxin-hemolysin (ACT) is a key virulence factor of the whooping cough agent Bordetella pertussis (Bp). The major cytotoxic activity of this 1706-residue protein consists of its capacity to invade a variety of eukaryotic cells directly across their cytoplasmic membrane and to deliver into cells a catalytic adenylate cyclase domain. This causes impairment of immune effector cells and apoptosis of lung macrophages by uncontrolled conversion of ATP to cAMP. The adenylate cyclase toxin-hemolysin acquires biological activity upon post-translational amide-linked palmitoylation of the epsilon-amino group of lysine 983 (K983) by the accessory fatty acyltransferase, CyaC. However, an additional conserved acylation site can be identified in ACT at lysine 860 (K860) and this residue is palmitoylated when recombinant ACT is produced in Escherichia coli (r-Ec-ACT). In this paper we report the double acylation of r-Bp-ACT secreted by a recombinant Bp strain 18323/pHSP9. This strain overproduces ACT from an oligocopy plasmid carrying the entire cya locus of Bordetella pertussis 18323. Palmitoylation of both conserved lysines (K860 and K983) of r-Bp-ACT expressed by this Bp strain was found. In addition, an error in the deduced protein sequence was identified, with Leu being the real residue at position 1001 and not the Val residue given in the published gene sequence. We also discuss these results in comparison with those from recombinant ACT expressed in E. coli strain K12 XL1-Blue. The analytical approach for characterization of the fatty acylation of ACT from strain 18323/pHSP9 consisted of multiple proteolytic digestion procedures (trypsin, Asp-N), microcapillary liquid chromatography/tandem mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

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The glucose and nitrogen starvation response of Bacillus licheniformis

et al. 2007

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The glucose and nitrogen starvation stimulons of Bacillus licheniformis were determined by transcriptome and proteome analyses. Under both starvation conditions, the main response of B. licheniformis was a switch to the usage of alternative nutrient sources. This was indicated by an induction of genes involved in the metabolism of C-2 substrates during glucose limitation. In addition, B. licheniformis seems to be using other organic substances like amino acids and lipids as carbon sources when subjected to glucose starvation. This observation is supported by the induction of a high number of genes coding for proteins involved in amino acid and lipid degradation. During nitrogen starvation, genes for several proteases and peptidases involved in nitrate and nitrite assimilation were induced, which enables this bacterium to recruit nitrogen from alternative sources. Both starvation conditions led to a down-regulation of transcription of most vegetative genes, which was subsequently reflected by a reduced synthesis of the corresponding proteins. A selected set of genes was induced by both starvation conditions. Among them were yvyD, citA and the putative methylcitrate shunt genes mmgD, mmgE and yqiQ. However, both starvation conditions did not induce a general SigmaB-dependent stress response.

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Analysis of the in vivo activation of hemolysin (HlyA) from Escherichia coli

Cited by 67 publications

References 48 publications

In Vivo Proteolytic Degradation of the Escherichia coli Acyltransferase HlyC

In Vivo Proteolytic Degradation of the Escherichia coli Acyltransferase HlyC

Mass spectrometric analysis of recombinant adenylate cyclase toxin from Bordetella pertussis strain 18323/pHSP9

The glucose and nitrogen starvation response of Bacillus licheniformis

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