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R C Goldman

Publications and source records attributed to R C Goldman.

At least 55 records · Page 3Linked to original sources
PubMed1988

Lipid A precursor from Pseudomonas aeruginosa is completely acylated prior to addition of 3-deoxy-D-manno-octulosonate.

Inhibition of lipopolysaccharide (LPS) synthesis in Pseudomonas aeruginosa at the stage of incorporation of 3-deoxy-D-manno-octulosonate (KDO) caused accumulation of a lipid A precursor which contained all of the fatty acids present on the lipid A of mature LPS. The enzyme CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase) from P. aeruginosa is inhibited by the KDO analog alpha-C-[1,5-anhydro-8-amino-2,7,8-trideoxy-D-manno-octopyranosyl] carboxylate (I), and I is effectively delivered to P. aeruginosa following attachment by amide linkage to the carboxyl terminus of alanylalanine. Intracellular hydrolysis releases the free inhibitor (I) which then inhibits activation of KDO by CMP-KDO synthetase causing accumulation of lipid A precursor and subsequent growth stasis. The major lipid A precursor species accumulated was purified and found to contain glucosamine, phosphate, C12:O, 2OH-C12:O and 3OH-C10:0 (in ester linkage), and 3OH-C12:0 (in amide linkage) in molar ratios of 1:1:0.5:0.5:1:1. Analysis of precursor by fast atom bombardment mass spectroscopy yielded a major ion (M - H)- of mass 1616 and fragments which were consistent with the structure of lipid A from P. aeruginosa. In contrast, Salmonella typhimurium, Escherichia coli, Citrobacter sp., Serratia marcescens, Enterobacter aerogenes, and Enterobacter cloacae all accumulated underacylated lipid A precursors which only contained 3-OH-C14:0, glucosamine, and phosphate. This difference and species-specific patterns of major and minor precursor species show that early steps in the assembly of lipid A are similar, but not identical in enteric and nonenteric Gram-negative bacteria.

Acetylglucosamine↗
PubMed1988

Analysis of lipopolysaccharide biosynthesis in Salmonella typhimurium and Escherichia coli by using agents which specifically block incorporation of 3-deoxy-D-manno-octulosonate.

Antibacterial agents which specifically inhibit CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase activity were used to block the incorporation of 3-deoxy-D-manno-octulosonate (KDO) into lipopolysaccharide. Lipopolysaccharide synthesis ceased, molecules similar in structure to lipid A accumulated, and bacterial growth ceased following addition of such agents to cultures of Salmonella typhimurium and Escherichia coli. Although four major species of lipid A accumulated in S. typhimurium, their kinetics of accumulation were different. The least polar of the major species was IVA [O-(2-amino-2-deoxy-beta-D-glucopyranosyl)-(1----6)-2-amino-2-deoxy-a lph a- D-glucose, acylated at positions 2, 3, 2', and 3' with beta-hydroxymyristoyl groups and bearing phosphates at positions 1 and 4'], a molecule previously isolated from bacteria containing a kdsA mutation (C. R. H. Raetz, S. Purcell, M. V. Meyer, N. Qureshi, and K. Takayama, J. Biol. Chem. 260:16080-16088, 1985). Species IVA accumulated first and to the greatest extent following addition of the inhibitor, with other more polar derivatives appearing only after IVA attained half its maximal level. In contrast, only two major species of precursor accumulated in E. coli following addition of the inhibitor. One of these species was identical to IVA from S. typhimurium on the basis of chemical composition, fast atom bombardment mass spectroscopy, and comigration on Silica Gel H, and it also accumulated prior to a more polar species of related structure. We conclude that the addition of KDO to precursor species IVA is the major pathway of lipid A-KDO formation in both S. typhimurium LT2 and E. coli and that accumulation of the more polar species lacking KDO only occurs in response to accumulation of species IVA following inhibition of the normal pathway.

Anti-Bacterial Agents↗
PubMed1987

Isolation of Salmonella typhimurium strains that utilize exogenous 3-deoxy-D-manno-octulosonate for synthesis of lipopolysaccharide.

Spontaneous mutants of Salmonella typhimurium LT2 were selected for the ability to accumulate exogenous 3-deoxy-D-manno-octulosonate (KDO). Bacteria containing a gene (kdsA) which codes for a temperature-sensitive KDO-8-phosphate synthetase were plated at the restrictive temperature of 42 degrees C on medium containing 5 mM KDO. Since bacteria containing the kdsA lesion are unable to grow at 42 degrees C due to inhibition of lipopolysaccharide (LPS) synthesis and accumulation of lipid A precursor, this method allowed direct, positive selection of mutants capable of utilizing exogenous KDO for LPS synthesis. Spontaneous mutants, selected at a frequency of about 10(-6), required exogenous KDO for growth at 42 degrees C. The growth rate at 42 degrees C was nearly normal in the presence of 20 mM KDO and was directly proportional to KDO concentrations below 20 mM. Exogenous KDO also suppressed accumulation of lipid A precursor. The apparent Km for KDO accumulation was 23 mM, and the maximum rate of transport was calculated to be 505 pmol of KDO per min per 10(8) cells. Bacteria incorporated exogenous [3H]KDO exclusively into LPS, with less than 10% dilution in specific activity due to residual endogenous KDO synthesis. The mutation giving rise to the ability to accumulate exogenous KDO was extremely useful in the direct screening for new mutations in the kdsA gene after localized mutagenesis. Five mutations in kdsA were isolated, four of which were new alleles as determined by on fine-structure analysis. The ability to introduce labeled (3H, 13C, and 14C) KDO in vivo should simplify and extend the analysis of this critical metabolic pathway in gram-negative bacteria.

DNA Transposable Elements↗
PubMed1987

Inhibition of exogenous 3-deoxy-D-manno-octulosonate incorporation into lipid A precursor of toluene-treated Salmonella typhimurium cells.

Analogs of 3-deoxy-D-manno-octulosonate (KDO) were designed to inhibit CTP:CMP-KDO cytidylyltransferase (CMP-KDO synthetase). Since these analogs lacked whole-cell antibacterial activity, a permeabilized-cell method was developed to measure intracellular compound activity directly. The method employed a mutant of Salmonella typhimurium defective in KDO-8-phosphate synthetase (kdsA), which accumulated lipid A precursor at 42 degrees C. Cells permeabilized with 1% toluene were used to evaluate inhibitor effect on [3H]KDO incorporation into preformed lipid A precursor. KDO incorporation proceeded through the enzymes CMP-KDO synthetase and CMP-KDO:lipid A KDO transferase. Optimum KDO incorporation occurred between pH 8 and 9 and required CTP, prior lipid A precursor accumulation, and a functional kdsB gene product, CMP-KDO synthetase. The apparent Km for KDO in this coupled system at pH 7.6 was 1.38 mM. The reaction products isolated and characterized contained 1 and 2 KDO residues per lipid A precursor molecule. Several KDO analogs produced concentration-related reductions of KDO incorporation in toluenized cells with 50% inhibitory concentrations comparable to those obtained in purified CMP-KDO synthetase systems. Two compounds, 8-amino-2-deoxy-KDO (A-60478) and 8-aminomethyl-2-deoxy-KDO (A-60821), competitively inhibited KDO incorporation, displaying Kis of 4.2 microM for A-60478 and 2.5 microM for A-60821. These data indicated that the inactivity of the KDO analogs on intact bacteria was the result of poor permeation into cells rather than intracellular inactivation.

Cell Membrane↗
PubMed1986

Primary structure of CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase) from Escherichia coli.

The gene coding for CTP:CMP-3-deoxy-D-mannooctulosonate cytidylyltransferase (CMP-KDO synthetase), kds B, was previously cloned on a 9-kilobase Pst insert of Escherichia coli DNA into pBR 322 (Goldman, R. C., and Kohlbrenner, W. E. (1985) J. Bacteriol. 163, 256-261). Using a transposon mutagenesis approach we have now located kds B on this insert, which facilitated the isolation and sequencing of a 1.3-kilobase segment of DNA containing kds B and putative RNA polymerase and ribosome binding sites. The primary structure of CMP-KDO synthetase predicted by this nucleotide sequence was verified by amino acid composition and sequence analysis of purified CMP-KDO synthetase and cleavage fragments. Our results show that kds B consists of a 744-base open reading frame coding for a 248-amino acid peptide. The molecular weight of CMP-KDO synthetase calculated from the translated sequence is 27,486, taking into account the loss of the N-terminal methionine. These data define the transcriptional unit of kds B and its translation product in molecular terms, information prerequisite to our understanding of both the mechanism of CMP-KDO formation and the regulation of the KDO metabolic pathway in Gram-negative bacteria.

Amino Acid Sequence↗
PubMed1985

Molecular cloning of the structural gene coding for CTP:CMP-3-deoxy-manno-octulosonate cytidylyltransferase from Escherichia coli K-12.

The kdsB gene from Escherichia coli K-12, which encodes CTP:CMP-3-deoxy-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase), was cloned into pBR322 as an 8-kilobase PstI fragment. Selection of this cloned segment was facilitated by using Salmonella typhimurium SL5283, which is deficient in three restriction enzyme systems and thus allows efficient cloning of E. coli DNA in S. typhimurium. The temperature-sensitive kdsB gene from S. typhimurium HD2 was transduced into strain SL5283 after the insertion of transposon Tn10 near the kdsB allele. Tetracycline-sensitive variants of strain SL5283 were then derived and used to select clones of the E. coli K-12 gene, inserted into the PstI site of pBR322, by complementation of the temperature-sensitive lesion in kdsB. One plasmid, pRG-1, complemented the kdsB temperature-sensitive allele and had the following characteristics: (i) it coded for several polypeptides by coupled transcription-translation in vitro, including one polypeptide which comigrated with CMP-KDO synthetase during polyacrylamide gel electrophoresis in sodium dodecyl sulfate; (ii) it overproduced CMP-KDO synthetase activity 20- to 40-fold depending on strain and growth conditions; and (iii) it coded for activity of CMP-KDO synthetase which, when purified to homogeneity, had the same molecular weight and kinetic characteristics as CMP-KDO synthetase of chromosomal origin.

Chromosome Mapping↗
PubMed1984

Mechanism of bacterial resistance to complement-mediated killing: inserted C5b-9 correlates with killing for Escherichia coli O111B4 varying in O-antigen capsule and O-polysaccharide coverage of lipid A core oligosaccharide.

The interaction of C3 and terminal complement components with three isogenic strains of Escherichia coli O111B4 varying in outer membrane and capsule composition was examined. Strains CL99 and 1-1, which possess O-antigen capsule and 74 to 77% coverage of lipid A core oligosaccharide, were sensitive to killing in pooled normal human serum (PNHS) or magnesium ethylene glycoltetraacetic acid PNHS in the presence but not the absence of antibody, although 1-1 contained 35% more lipopolysaccharide than CL99 and was slightly less sensitive to alternative pathway killing. In contrast, strain 1-2 lacks O-antigen capsule but contains 84% coverage and resists serum killing in the presence and absence of antibody in both PNHS and magnesium ethylene glycoltetraacetic acid PNHS. All three strains consumed C3 and C9 when incubated in PNHS, but consumption was most rapid with 1-2, which also bound the largest number of C3 molecules per CFU. Between 15 X 10(3) and 24 X 10(3) molecules of C9 per CFU bound to CL99 and 1-1 during incubation in 10% PNHS or 10% magnesium ethylene glycoltetraacetic acid PNHS, and binding was relatively stable. Binding and release of 3 X 10(3) to 8 X 10(3) molecules of C9 per CFU was observed for strain 1-2. The majority of C9 bound to CL99 and 1-1 in the presence of antibody distributed with the outer membrane after lysis of the organisms in a French press, whereas only 16.1 to 20.1% of C9 was deposited on these organisms in the absence of antibody, and 31.5 to 39.8% of C9 on strain 1-2 with or without antibody sedimented with the outer membrane. Between 4.6 X 10(3) and 5.5 X 10(3) molecules of C9 per CFU remained bound in a salt- and trypsin-resistant form to the outer membrane of organisms that were killed, whereas fewer than 1.4 X 10(3) molecules of C9 per CFU were bound to the outer membrane of organisms not killed by serum. These results indicate that C5b-9 that is bound to the outer membrane of E. coli O111B4 in a form resistant to salt or protease elution correlates with bacterial killing.

Antigens, Bacterial↗
PubMed1984

Serum-resistant mutants of Escherichia coli O111 contain increased lipopolysaccharide, lack an O antigen-containing capsule, and cover more of their lipid A core with O antigen.

Escherichia coli strains of group O111 were characterized with respect to sensitivity to complement killing, amount of lipopolysaccharide and O antigen-containing capsule, and distribution of O antigen. All wild-type E. coli O111 strains were resistant to complement killing in the absence of specific antibody. Presensitization of strains with antibody to whole cells (OK antibody), followed by incubation in 50% pooled normal human serum as a source of complement, subdivided wild-type strains into three types: completely resistant, partially resistant, and sensitive. Completely and partially resistant mutants were isolated by cycles of serum killing, starting with one sensitive strain. Completely resistant mutants had no O antigen-containing capsule, but had 50% more lipopolysaccharide than did the parent, and this lipopolysaccharide had 30% fewer lipid A core molecules devoid of O antigen. Partially resistant mutants still had O antigen-containing capsule, but contained 40% more lipopolysaccharide than did the parent; the extent of coverage of lipid A core with O antigen remained unchanged. No correlations were found between outer membrane protein composition and the degree of serum resistance. Since the terminal membrane attack complex (C5b-9) must stably insert into a hydrophobic membrane site to effect killing, we conclude that both increased lipid A core and increased coverage of lipid A core with O antigen preclude access of C5b-9 to lethal sites on the cell surface.

Antigens, Bacterial↗
PubMed1983

Quantitative double-label radiography of two-dimensional protein gels using color negative film and computer analysis.

We have devised a method of data collection and computer analysis which allows utilization of the resolving power of two-dimensional gel electrophoresis of proteins, in conjunction with the versatility of using two different radionuclides simultaneously. Cultures of Escherichia coli growing with exponential growth rate constants (mu) of 0.32 and 1.43 were labeled with [3H]leucine and [14C]leucine, respectively; these samples were mixed, and cell protein was separated on a two-dimensional gel. Spacial and quantitative data for both radionuclides were recorded on color negative film by radiographic exposure. Data for 14C alone were then collected photographically from the red-light-sensitive layer of the film using a red filter, while data for 3H and spillover of 14C were collected photographically from the blue-light-sensitive layer using a blue filter. These two data sets were analyzed by CINT, a computer program for analysis of two-dimensional gels, and quantitative data for 3H were calculated after determination of spillover of 14C in a manner analogous to quantification of 3H and 14C by liquid scintillation counting. Quantitative data from over 1000 protein spots representing from 0.002% to 10% of the total 3H or 14C, respectively, are available in a matter of hours. We have used this method to analyze the effect of growth rate and medium composition on the relative levels of individual proteins in a pathogenic strain of E. coli which contains group 111 O-antigen. As expected, the relative levels of aminoacyl-tRNA synthetases, protein chain elongation factors, ribosomal proteins, and the alpha-subunit of RNA polymerase are all increased with increased growth rate; the magnitude of these changes agreed with previous data derived using other strains of E. coli. Alterations in the levels of other proteins identified on the two-dimensional gels could be interpreted in terms of changes in medium composition. When compared to manual data collection by excising radiolabeled proteins and quantifying 3H and 14C in a liquid scintillation counter following combustion to H2O and CO2, respectively, this new method of data collection and computer analysis increases the resolution of data collection and decreases the time involved from days to hours.

Bacterial Proteins↗
PubMed1983

Studies of the mechanism of bacterial resistance to complement-mediated killing. V. IgG and F(ab')2 mediate killing of E. coli 0111B4 by the alternative complement pathway without increasing C5b-9 deposition.

The mechanism of antibody-dependent complement-(C) mediated killing of Escherichia coli 0111B4, strain 12015 (12015), was examined. 12015 was resistant to serum killing when incubated in hypogammaglobulinemic serum (H gamma S) or pooled normal human serum (NHS) that had been previously adsorbed to remove specific antibody (Abs NHS). Presensitization with immune rabbit serum or purified immune rabbit IgG resulted in 1 to 3 log killing when 5 X 10(8) colony forming units (CFU)/ml were incubated in 10 to 40% Abs NHS. Binding of 125I-C3 and 131I-C9 to the bacterial surface of the presensitized and the nonpresensitized strain was quantitated when these organisms were incubated in 10, 20, and 40% Abs NHS. Stable binding of up to 3.0 X 10(5) molecules of C3 and 8.0 X 10(4) molecules of C9 to presensitized and nonpresensitized isolates occurred in the highest concentration of serum, but there was no killing without presensitization. Similar results were found when Abs NHS was chelated with ethylene bis glycoltetraacetic acid containing 2 mM MgCl2 (Mg EGTA) to block classical pathway activation, indicating that antibody mediated the bactericidal reaction through the alternative pathway. Deposition of C3 and C9 and killing of 120 15 in 10% Abs NHS or 10% H gamma S was measured after presensitization with increasing amounts of IgG, F(ab')2, or Fab'. There was a dose-dependent increase in C3 deposition and killing, but only minimal change in C9 binding when 1.0 X 10(3) to 3.2 X 10(4) IgG or F(ab')2/CFU were bound to the bacterial surface. In contrast, there was no increase in C3 or C9 binding and no bacterial killing when 1 X 10(3) to 3.4 X 10(4) molecules Fab'/CFU were bound to the bacterial surface. These experiments show that immune IgG and F(ab')2 can mediate killing of E. Coli 0111B4 by the alternative pathway without changing the extent of terminal C component attachment to the bacterial surface.

Animals↗
PubMed1983

Studies on the mechanism of bacterial resistance to complement-mediated killing. VI. IgG increases the bactericidal efficiency of C5b-9 for E. coli 0111B4 by acting at a step before C5 cleavage.

Our previous experiments showed that immune IgG and F(ab')2, but not Fab', mediated serum killing of Escherichia coli 0111B4, strain 12015 (12015), without significantly increasing the extent of terminal complement (C) component attachment to the bacterial surface. We concluded that bactericidal antibody must change either the site or the nature of C5b-9 bacterial attachment. To pursue this possibility, conditions necessary for elution of C5b-9 from the bacterial surface were examined. Forty-two to 44% of 125I-C9 was released from the serum-resistant nonpresensitized 12015 by 1 M NaCl or 0.1% trypsin, compared with the 21 to 24% release from the serum-sensitive presensitized isolate under the same condition. When strain 12015 bearing 125I-C9 was lysed in a French pressure cell, 73.1% of 125I-C9 was released with the capsular fraction if the organisms had not been presensitized. In contrast, on presensitized 12015, 70.2% of 125I-C9 remained associated with the outer membrane after such lysis. These results suggested that C5b-9 was trapped within or underneath the capsule of 12015 in the absence of bactericidal antibody, but that addition of antibody led to C5b-9 insertion into the outer membrane with bacterial killing. The requirement of C components preceding C5 for bacterial killing was next examined. Minimal killing of presensitized 12015 occurred when a terminal C complex was formed by acid activation from purified C5, C6, C7, C8, and C9 in the absence of C3 or earlier components. In contrast, between 1.2 and 3 log killing of nonpresensitized rough Salmonella minnesota and rough E. coli was observed in the same system. Killing of 12015 was examined with bacteria incubated in C5-deficient serum (C5D), followed by washing and the addition of purified C5, C6, C7, C8, and C9 to permit C5b-9 formation. Antibody was added before or after incubation in C5D serum, or after the addition of purified C5-C9. Under conditions of equivalent C3 and C9 binding, significant killing occurred only when antibody was added before incubation in C5D serum. These results show that antibody must be present at or before the time of C5 convertase formation to mediate killing of 12015 by C5b-9. Therefore, antibody is unlikely to be functioning primarily to alter the bacterial surface to expose sites for C5b-9 insertion, nor is the effect of antibody simply to increase C3 and terminal component binding. We postulate that antibody mediates killing of 12015 by localizing C5b-9 around antibody-clustered sites of C3 and C5 convertase formation.

Animals↗
PubMed1982

A surface polysaccharide of Escherichia coli O111 contains O-antigen and inhibits agglutination of cells by O-antiserum.

The repeating pentasaccharide of O-antigen from Escherichia coli O111 contains galactose, glucose, N-acetylglucosamine, and colitose, the latter representing the major antigenic determinant. Phenol extraction of this strain was previously shown to release two fractions (I and II) containing O-antigen carbohydrate, and both fractions were believed to be lipopolysaccharide. We have now characterized fractions I and II and conclude that only fraction II represents lipopolysaccharide. Fraction II contains phosphate, 2-keto-3-deoxyoctonate, beta-hydroxymyristic acid, and potent endotoxin activity, whereas fraction I was deficient in all of these properties of the lipid A and core oligosaccharide regions of lipopolysaccharide. Fractions I and II each represented 50% of the total cellular O-antigen, and both were present on the cell surface. Both fractions were metabolically stable, and no precursor-product relationship existed between them. Fraction II had a number-average molecular weight of 15,800, corresponding to an average of 12 O-antigen repeats per molecule. In contrast, fraction I had a number-average molecular weight of 354,000, corresponding to an average of 404 O-antigen repeats per molecule. Before heat treatment, cells of E. coli O111 are poorly agglutinated by O-serum; although this indicates the presence of a capsule, the corresponding K-antigen was never detected. We conclude that fraction I, when present on the cell surface, inhibits agglutination of unheated cultures of E. coli O111 by O-serum because: (i) a variant strain which lacks fraction I was agglutinated by O-serum without prior heating; (ii) erythrocytes coated with purified fraction I behaved like bacteria containing fraction I in showing inhibition of O-serum agglutination; and (iii) heat treatment released fraction I and rendered bacterial cells agglutinable in O-serum.

Agglutination↗
PubMed1981

Coat protein synthesis during sporulation of Bacillus subtilis: immunological detection of soluble precursors to the 12,200-dalton spore coat protein.

Antibody specific to the 12,200-dalton spore coat protein of Bacillus subtilis was used to detect the synthesis of cross-reacting material during sporulation. Cross-reacting protein was first detected by immunoprecipitation after 4 h of development and represented at least 1 to 2% of the total soluble protein synthesis at 5.5 h. A polypeptide of 21,000 daltons was detected in immunoprecipitates by gel electrophoresis. This polypeptide did not accumulate in sporulating cells and was rapidly turned over at the time of coat deposition. In contrast, a 32,000-dalton polypeptide reacted with antibody when unlabeled cell protein was denatured with sodium dodecyl sulfate, separated by gel electrophoresis, and transferred to nitrocellulose paper. This polypeptide was not detected during cell growth or the first 3.5 h of development but was found to accumulate in sporulating cells at 5.5 h. The lack of detection of this polypeptide by immunoprecipitation of undenatured protein indicates that the antigenic sites which cross-reacted with antibody to the 12,200-dalton protein sequence were not exposed unless the molecular conformation was altered. The 32,000-dalton protein may be a primary translation product which is proteolytically processed into mature spore coat protein via a 21,000-dalton intermediate.

Bacillus subtilis↗
PubMed1979

Morphology and patterns of protein synthesis during sporulation of Bacillus subtilis Eryr spo(Ts) mutants.

Erythromycin-resistant (Eryr) mutants of Bacillus subtilis 168 fail to sporulate at high temperature (47 degrees C) but sporulate normally at 30 to 35 degrees C. They also fail to sporulate at any temperature in the presence of 2.5 micrograms of erythromycin per ml. Neither of these nonpermissive conditions appears to affect vegetative growth, and the periods of sensitivity to both conditions extend from 40 to 90% of the sporulation period. At 47 degrees C, net incorporation of methionine and phenylalanine in postexponential Eryr and 168 cells was similar, and fractionation of the labeled products by polyacrylamide gel electrophoresis gave patterns in which many of the bands produced by mutant and parental cells coincided. However, distinct differences were seen, and since no spore-specific morphogenesis occurred in the Eryr cells at 47 degrees C, a selective defect in spore gene expression was inferred. At 35 degrees C plus erythromycin, spore morphogenesis proceeded normally until forespores were produced and then ceased, coincident with a marked increase in sensitivity of total protein synthesis to erythromycin. The effects seem to be nonspecific, therefore, and may indicate a change in cell permeability or ribosomal sensitivity to erythromycin.

Bacillus subtilis↗
PubMed1978

Bacillus subtilis spore coats: complexity and purification of a unique polypeptide component.

Extensively washed, dormant spores of Bacillus subtilis were disrupted with glass beads in buffer at pH 7 in the presence of protease inhibitors. Approximately 31% of the total spore protein was soluble, and another 14% was removed from the insoluble fraction by hydrolysis with lysozyme and washing with 1 M KCl and 0.1% sodium dodecyl sulfate. The residual spore integuments comprised 55% of the total spore proteins and consisted of coats and residual membrane components. Treatment of integuments with sodium dodecyl sulfate and reducing agents at pH 10 solubilized 40% of the total spore protein. Seven low-molecular-weight polypeptide components of this solubilized fraction comprised 27% of the total spore protein. They are not normal membrane components and reassociated to form fibrillar structures resembling spore coat fragments. The residual insoluble material (15% of the total spore protein) was rich in cysteine and was probably also derived from the spore coats. A solubilized coat polypeptide of molecular weight 12,200 has been purified in good yield (4 to 5% of the total spore protein). Five amino acids account for 92% of its total amino acid residues: glycine, 19%; tyrosine, 31%; proline, 23%; arginine, 13%; and phenylalanine, 6%.

Amino Acids↗