Prevalence of temperature sensitive folding mutations in the parallel beta coil domain of the phage P22 tailspike endorhamnosidase

Haase-Pettingell, Cameron; King, Jonathan

doi:10.1006/jmbi.1996.0841

Cited by 44 publications

(47 citation statements)

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“…The temperature sensitivity of SMEG35 could result from thermolability of the mutant protein, a general folding defect (as described previously for EcoRI endonuclease and phage P22 tail spike endorhamnosidase [9,15]), or a general reduction in specific activity resulting in a mutant protein that cannot keep up with the increased metabolic demands of growth at higher temperatures. To address this, the wild-type and mutant DdlA proteins were purified and tested for enzymatic activity at different temperatures.…”

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Genetic Analysis of Peptidoglycan Biosynthesis in Mycobacteria: Characterization of a ddlA Mutant of Mycobacterium smegmatis

2000

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A temperature-sensitive mutant of Mycobacterium smegmatis was characterized that contains a mutation in ddlA, the gene encoding D-alanine:D-alanine ligase. Enzymatic assays using recombinant proteins and Dcycloserine susceptibility indicate that the A365V mutation in the SMEG35 DdlA protein causes a reduction in enzymatic activity in vitro and in vivo.A nearly universal component of bacterial cell walls is peptidoglycan, a macromolecule that is composed of polysaccharide chains that are cross-linked by short peptide bridges. Peptidoglycan gives the bacterial cell its characteristic shape and prevents the cell from lysing due to high internal osmotic pressure. Our understanding of how peptidoglycan is synthesized in bacteria is derived mostly from work done with Escherichia coli in which a number of temperature-sensitive mutants have been isolated that are defective in the biosynthesis of peptidoglycan at 42°C (10,11,13,14,20). Two hallmarks of these mutants are cell lysis at the nonpermissive temperature and suppression of the temperature-sensitive phenotype on media containing osmotic stabilizers (10,11,13,14,20).We previously described the generation of a bank of temperature-sensitive mutants of Mycobacterium smegmatis mc 2 155 (2, 4). One of the mutants, SMEG35, exhibits the two phenotypic characteristics associated with E. coli mutants defective in peptidoglycan biosynthesis. First, SMEG35 cells grown at 30°C to an optical density at 600 nm of 0.5 and then shifted to 42°C lyse after one doubling time, as evidenced by a visual clearing of the culture and the appearance of flocculent material (data not shown). Second, the temperature-sensitive phenotype of SMEG35 can be suppressed on growth medium containing either 0.5 M sucrose or 0.2 M NaCl (data not shown).The bacterial strains and plasmids used in this study are listed in Table 1. To identify the mutated gene, SMEG35 was complemented with an M. smegmatis genomic cosmid library as previously described (2). A sublibrary was constructed from the complementing cosmid pAEB222 and the E. coli-mycobacterial shuttle vector pMD30 as described previously (2). The nucleotide sequence was determined for the insert of the smallest complementing subclone pAEB224 with an ABI310 automated sequencer (PE Biosystems, Foster City, Calif.) and genespecific primers. The presence of a mutation in the ddlA gene of SMEG35 was confirmed by sequencing of the mutant allele. The gene was amplified from the SMEG35 genome with Pfu DNA polymerase (Stratagene) and the primers 5Ј-TTGTGACTGCCCC GAACC-3Ј (forward) and 5Ј-CGAAAAACCCGTCGAGCC-3Ј (reverse) in a PCR mixture supplemented with 5% formamide. Sequence analysis revealed a single mutation at bp 1095 of ddlA that changes a C to a T on the top strand. This mutation results in an alanine-to-valine substitution at amino acid 365 of Ddl, close to the C terminus of the protein. The alanine that is mutated in the SMEG35 Ddl is a nonconserved amino acid residue that does not correspond to any of the amino acids that were previously shown to be...

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Genetic Analysis of Peptidoglycan Biosynthesis in Mycobacteria: Characterization of a ddlA Mutant of Mycobacterium smegmatis

2000

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“…This suggested that the high-temperature-related problem was not failure of the assembly of native tailspike onto phage heads. Although native tailspike is thermostable, the presence of a thermolabile intermediate in the folding pathway has been well documented (21,22). To examine chain folding and assembly in vivo, we used a P22 strain [2 H200 (Am)/13 H101 (Am) C 1 7] containing an amber mutation in the DNA-packaging protein encoded by gene 2 (1).…”

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confidence: 99%

“…aggregates into an inclusion body if folding occurs at high temperatures (21,22) and prevents the production of native tailspike (Fig. 1A).…”

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confidence: 99%

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Protein Folding Failure Sets High-Temperature Limit on Growth of Phage P22 in Salmonella enterica Serovar Typhimurium

Pope

Haase-Pettingell

King

2004

Appl Environ Microbiol

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The high-temperature limit for growth of microorganisms differs greatly depending on their species and habitat. The importance of an organism's ability to manage thermal stress is reflected in the ubiquitous distribution of the heat shock chaperones. Although many chaperones function to reduce protein folding defects, it has been difficult to identify the specific protein folding pathways that set the high-temperature limit of growth for a given microorganism. We have investigated this for a simple system, phage P22 infection of Salmonella enterica serovar Typhimurium. Production of infectious particles exhibited a broad maximum of 150 phage per cell when host cells were grown at between 30 and 39°C in minimal medium. Production of infectious phage declined sharply in the range of 40 to 41°C, and at 42°C, production had fallen to less than 1% of the maximum rate. The host cells maintained optimal division rates at these temperatures. The decrease in phage infectivity was steeper than the loss of physical particles, suggesting that noninfectious particles were formed at higher temperatures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a decrease in the tailspike adhesins assembled on phage particles purified from cultures incubated at higher temperatures. The infectivity of these particles was restored by in vitro incubation with soluble tailspike trimers. Examination of tailspike folding and assembly in lysates of phage-infected cells confirmed that the fraction of polypeptide chains able to reach the native state in vivo decreased with increasing temperature, indicating a thermal folding defect rather than a particle assembly defect. Thus, we believe that the folding pathway of the tailspike adhesin sets the high-temperature limit for P22 formation in Salmonella serovar Typhimurium.

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“…1995). All of the tsf mutations occur in the center of gene 9 between residues 150 and 500 in the &coil domain (Villafane & King, 1988: Haase-Pettingell & King, 1997. Structural analysis of the native tailspike suggests that tsf mutations may cause the misfolding of the p-coil domain in the monomeric folding intermediates (King et al, 1996).…”

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Conformation of P22 tailspike folding and aggregation intermediates probed by monoclonal antibodies

Speed¹,

Morshead²,

Wang³

et al. 1997

Protein Science

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The partitioning of partially folded polypeptide chains between correctly folded native states and off-pathway inclusion bodies is a critical reaction in biotechnology. Multimeric partially folded intermediates, representing early stages of the aggregation pathway for the P22 tailspike protein, have been trapped in the cold and isolated by nondenaturing polyacrylamide gel electrophoresis (PAGE) (Speed MA, Wang DIC, King J. 1995. Protein Sci 4:900-908). Monoclonal antibodies against tailspike chains discriminate between folding intermediates and native states (Friguet B, DjavadiOhaniance L, King J, Goldberg ME. 1994. J Biol Chem 269:15945-15949). Here we describe a nondenaturing Western blot procedure to probe the conformation of productive folding intermediates and off-pathway aggregation intermediates. The aggregation intermediates displayed epitopes in common with productive folding intermediates but were not recognized by antibodies against native epitopes. The nonnative epitope on the folding and aggregation intermediates was located on the partially folded N-terminus, indicating that the N-terminus remained accessible and nonnative in the aggregated state. Antibodies against native epitopes blocked folding, but the monoclonal directed against the N-terminal epitope did not, indicating that the conformation of the N-terminus is not a key determinant of the productive folding and chain association pathway.

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Prevalence of temperature sensitive folding mutations in the parallel beta coil domain of the phage P22 tailspike endorhamnosidase

Cited by 44 publications

References 73 publications

Genetic Analysis of Peptidoglycan Biosynthesis in Mycobacteria: Characterization of a ddlA Mutant of Mycobacterium smegmatis

Genetic Analysis of Peptidoglycan Biosynthesis in Mycobacteria: Characterization of a ddlA Mutant of Mycobacterium smegmatis

Protein Folding Failure Sets High-Temperature Limit on Growth of Phage P22 in Salmonella enterica Serovar Typhimurium

Conformation of P22 tailspike folding and aggregation intermediates probed by monoclonal antibodies

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