A simple method for assigning multiple immunogens to their protein on a two-dimensional blot and its application to asthma-causing allergens.

A "one-step" procedure, that not only removes the color and blocking proteins used in the colorimetric immunodetection step but also stains the proteins originally on the blot, is presented. Following immunostaining and recording of immunoreactive spots, the blot was allowed to air-dry overnight (or longer) at room temperature and then counterstained with a colloidal gold solution. This "air-drying" process apparently altered the affinity of the blocking proteins (and possibly other proteins added subsequently to the blotting step) towards the nitrocellulose membrane causing them to be removed by the acidic colloidal gold solution while the "blotted" proteins were being stained. The sensitivity of this counterstained blot was comparable to that of the blot without going through the immunodetection process. Since both immunodetection and protein staining were carried out on the same blot, this allowed easy identification of many immunoreactive spots to their corresponding proteins when the two profiles were superimposed. Using this procedure, we have detected 25 immunoreactive spots (or allergens) from the whole body extract of the German cockroach (Blattella germanica) that contribute to asthma and assigned them to their corresponding proteins on a two-dimensional (2-D) protein map. The apparent Mr and pI for each of the allergens were determined. We have also located one of the major cockroach allergens, Bla g 5 (glutathione S-transferase). Two-dimensional zymography revealed the presence of ten gelatinase-type proteolytic enzymes. Only one of the ten proteases comigrated with the immunoreactive proteins indicating that unlike other allergen-producing systems, most of the cockroach allergens do not possess protease activity.

A new broad-spectrum protease inhibitor from the entomopathogenic bacterium Photorhabdus luminescens.

A new protease inhibitor was purified to apparent homogeneity from a culture medium of Photorhabdus luminescens by ammonium sulfate precipitation and preparative isoelectric focusing followed by affinity chromatography. Ph. luminescens, a bacterium symbiotically associated with the insect-parasitic nematode Heterorhabditis bacteriophora, exists in two morphologically distinguishable phases (primary and secondary). It appears that only the secondary-phase bacterium produces this protease inhibitor. The protease inhibitor has an M:(r) of approximately 12000 as determined by SDS-PAGE. Its activity is stable over a pH range of 3.5-11 and at temperatures below 50 degrees C. The N-terminal 16 amino acids of the protease inhibitor were determined as STGIVTFKND(X)GEDIV and have a very high sequence homology with the N-terminal region of an endogenous inhibitor (IA-1) from the fruiting bodies of an edible mushroom, Pleurotus ostreatus. The purified protease inhibitor inactivated the homologous protease with an almost 1:1 stoichiometry. It also inhibited proteases from a related insect-nematode-symbiotic bacterium, Xenorhabdus nematophila. Interestingly, when present at a molar ratio of 5 to 1, this new protease inhibitor completely inactivated the activity of both trypsin and elastase. The activity of proteinase A and cathepsin G was partially inhibited by this bacterial protease inhibitor, but it had no effect on chymotrypsin, subtilisin, thermolysin and cathepsins B and D. The newly isolated protease inhibitor from the secondary-phase bacteria and its specific inhibition of its own protease provides an explanation as to why previous investigators failed to detect the presence of protease activity in the secondary-phase bacteria. The functional implications of the protease inhibitor are also discussed in relation to the physiology of nematode-symbiotic bacteria.

Deficiency of current methods in assaying endochitinase activity.

Since chitin is degraded by a combination of both endo- and exochitinases, it is likely that both enzymes will be present in a crude extract. Currently used substrates for detecting endochitinase activity suffer from the fact that they could easily be digested by the contaminating exochitinase, thus giving either a false-positive or an inaccurate reading of the endochitinase activity. Using Photorhabdus luminescens, a bacterium symbiotically associated with insect-parasitic nematode Heterorhabditis bacteriophora as an exemplary system, we have identified these two chitinases by a simple "fluorimetric zymography" procedure. The exochitinase is a metalloenzyme and its activity is inhibited by 1,10-phenanthroline. Once the exochitinase activity is detected in a crude extract, its contribution must be eliminated before accurate determination of the endochitinase activity can be carried out. Specific properties of these enzymes including the pH activity profile, the requirement of metal ions for activity, and the molecular weight of the enzymes are among the factors to be considered in developing assaying procedures for endochitinase.

Analysis of proteins from different phase variants of the entomopathogenic bacteria Photorhabdus luminescens by two-dimensional zymography.

Two-dimensional zymography which combines two-dimensional electrophoresis with zymography was used to analyze proteases and other proteins produced by different phase variants of two strains of Photorhabdus luminescens. Both the primary and secondary phases of P. luminescens strains Hp and Hm secreted proteases. The protease in P. luminescens Hp has a molecular weight (Mr) of 57,000 and an isoelectric point (pI) of 4.4 whereas that in P. luminescens Hm has an Mr of 59,000 and pI of 4.9. Several putative protease degradation products were clearly visible in the zymograms from both bacterial strains. Two-dimensional zymography also showed that several secretory proteins were present only in particular phase variants and therefore could be used as specific markers. Unexpectedly, the two-dimensional zymography revealed that a nonsecretory protease with an Mr of 47,000 and a pI of 4.0 was present in the cell extracts of all phases of both P. luminescens Hp and Hm. The application of the two-dimensional zymography for the identification of other enzymes was also discussed.

Antimicrobial activity and biosynthesis of indole antibiotics produced by Xenorhabdus nematophilus.

We have investigated the mechanism of action and physiology of production of the indole derivative antibiotics produced by the nematode-associated, entomopathogenic bacterium Xenorhabdus nematophilus. Maximum antibiotic concentration was reached during the late stationary phase of growth, and the antibiotic yield was appreciably enhanced by supplementation with tryptophan. Antibiotic biosynthesis apparently involved the removal of the side-chain carboxyl (C-1) carbon of tryptophan. The C-3 methylene carbon of tryptophan, on the other hand, was retained. The purified indole antibiotic was effective against both Gram-positive and Gram-negative bacteria at low to moderate concentrations causing a severe inhibition of RNA synthesis, accompanied by a less severe effect on protein synthesis. An isogenic pair of Escherichia coli strains differing at the relA locus was used to demonstrate that the swift reduction in total RNA synthesis is related to an antibiotic-induced accumulation of the regulatory nucleotide, ppGpp, in susceptible bacteria. The E. coli relA mutant, which does not exhibit any discernible increase in ppGpp upon antibiotic treatment, showed no decrease in growth or RNA synthesis. Using this antibiotic, it was also observed that ppGpp may be employed as a metabolic regulator in bacteria such as Pseudomonas putida, which have not previously been reported to employ ppGpp as a regulatory molecule. We propose that the indole derivative antibiotic exerts growth inhibitory control in susceptible bacteria by greatly enhancing synthesis of ppGpp, resulting in a rapid inhibition of RNA synthesis.

The role of guanosine-3',5'-bis-pyrophosphate in mediating antimicrobial activity of the antibiotic 3,5-dihydroxy-4-ethyl-trans-stilbene.

The mode of action of 3,5-dihydroxy-4-ethyl-trans-stilbene (ES), an antibiotic produced by Xenorhabdus luminescens symbiotically associated with an entomopathogenic nematode, was investigated. ES was active against gram-positive and a number of gram-negative bacteria. In susceptible bacteria this antibiotic caused the inhibition of total RNA synthesis and, to a lesser extent, protein synthesis. At or above MICs, ES triggered a substantial accumulation of an intracellular regulatory compound, guanosine-3',5'-bis-pyrophosphate (ppGpp). This response was also noticed in species of bacteria which have previously not been shown to use ppGpp as a regulatory molecule. The involvement of ppGpp in antibiotic action was confirmed by using an isogenic stringent and a relaxed pair of Escherichia coli strains. The fact that the accumulation of ppGpp was correlated with the susceptibility of various gram-positive and gram-negative bacteria to ES suggests that this nucleotide is involved in the regulation of RNA synthesis and growth in all these microorganisms. Thus, inhibition of RNA synthesis via an increase in ppGpp concentrations may represent a mechanism that is prevalent among most bacteria and one that could be exploited for achieving a rapid inhibition of bacterial growth.

Processing of precursor ribosomal RNA and the presence of a modified ribosome assembly scheme in Escherichia coli relaxed strain.

An electrophoretic system capable of separating 25 S, 23 S, 17.5 S and 16 S ribosomal RNA (rRNA) species was used to study the synthesis and fate of rRNA during amino acid starvation and resupplementation of E. coli relaxed strain KL99. This E. coli relAl strain responded to an amino acid starvation by increasing the rate of synthesis of 25 S and 17.5 S precursor rRNA. When the limiting amino acid was resupplemented, a previously observed 40-fold increase in the cellular guanosine 5'-diphosphate, 3'-diphosphate content [Mol. Gen. Genet. (1983) 192, 5-9] appeared to cause a reduction in new rRNA synthesis. Following amino acid resupplementation, the precursor 25 S and 17.5 S rRNA accumulated during the amino acid starvation were conserved and processed to 23 S and 16 S rRNA species, respectively. This suggests that a modified ribosome assembly scheme involving stable precursor rRNA exists in relAl bacteria during periods of amino acid limitation and resupplementation.

Effect of ppGpp on the accuracy of protein biosynthesis.

The maintenance of accuracy in protein biosynthesis in amino acid-starved rel+ strains of Escherichia coli has been attributed to an effect of ppGpp on the accuracy of aa-tRNA selection by the ribosome. It has been determined that concentrations of ppGpp characteristic of those found in amino acid-starved cells have no effect on the rate of reaction of poly(U)-programmed ribosomes with either the cognate (Phe) or the near-cognate (Leu2) ternary complexes. Neither the rate of GTP hydrolysis, which signals selection of the ternary complex, nor the rate of peptide formation, which signals the acceptance of the aa-tRNA after proofreading, is affected by the nucleotide. The results indicate that the effect of ppGpp in maintaining the accuracy of protein biosynthesis in cells starved for an amino acid is not due to a direct effect on the rate constants for substrate selection by the ribosome.

Correlation between RNA synthesis and ppGpp content in Escherichia coli during temperature shifts.

Both a correlation and a lack of correlation between guanosine 5'-diphosphate, 3'-diphosphate (ppGpp) level and RNA accumulation have been reported during temperature shifts of E. coli. We have reexamined these phenomena by measuring the total rate of RNA synthesis. After a temperature upshift (23 degrees to 40 degrees C) of E. coli relA+ and relA1 strains, there is an immediate increase in the rate of RNA synthesis which corresponds with the observed in vitro effects of temperature on RNA synthesis (Mangel 1974; Travers 1974). A subsequent increase in ppGpp level is correlated with a decrease in the rate of RNA synthesis. Conversely, following a temperature downshift (40 degrees to 23 degrees C), both relA+ and relA1 bacteria show an immediate decrease in the rate of RNA synthesis. Subsequently all strains studied decrease ppGpp content and correspondingly increase the rate of RNA synthesis after a downshift. By measuring the rate of RNA synthesis we have separated immediate temperature-induced changes in RNA synthesis, from the apparent effects of ppGpp during temperature shifts. As a result, during temperature upshifts and downshifts of relA+, and relA1 bacteria, an inverse correlation between ppGpp content and the total rate of RNA synthesis does exist. The fact that both relA+ and relA1 strains show similar responses to temperature shifts provides additional evidence for the function of relA-independent basal level ppGpp synthesis in regulating RNA synthesis in E. coli.

Correlation between RNA synthesis and basal level guanosine 5'-diphosphate 3'-diphosphate in relaxed mutants of Escherichia coli.

Through the use of a new nucleotide extraction procedure, we had previously shown that relaxed mutants of Escherichia coli exhibit a unique response to amino acid starvation (Lagosky, P. A., and Chang, F. N. (1980) J. Bacteriol. 144, 499-508). The basal level amounts of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) in both relA and phenotypically relaxed relA+ rplK (relC) strains were shown to decrease at the onset of amino acid limitation and to remain severely depressed throughout the course of the starvation. Upon resupplementation of amino acid-starved relaxed mutants, the production of ppGpp resumes and results in the temporary overaccumulation of this nucleotide beyond its original basal level amount. We now show that the basal level ppGpp content of relaxed bacteria, as well as its subsequent fluctuations in response to amino acid starvation, is inversely correlated with the initial rates of RNA synthesis in these strains. The ability of ppGpp to control the rate of protein synthesis in relA mutants was also examined. It was observed that ppGpp had no apparent direct effect on the initial rates of protein synthesis in relA mutants. The constant inverse correlation which exists between ppGpp content in relA mutants, and their rates of RNa synthesis provide evidence which indicates that basal level ppGpp synthesis has definite physiological significance. It also suggests that the synthesis of basal level ppGpp might be an absolute requirement needed for normal bacterial growth.

Methylation of ribosomal proteins during ribosome assembly in Escherichia coli.

In Escherichia coli, a number of ribosomal proteins are methylated. The time of methylation of L7 and L11 during ribosome assembly was studied. It was observed that the methylation of L7 could occur in the free protein stage. Both the 32S and 40S ribonucleoprotein intermediates also contained methylated L7 although the extent of methylation in these particles was not as high as in the free L7, the 45S or the 50S particles. Free L11 could also be partially methylated but the bulk of methylation of this protein was found in the 45S and the 50S particles. It was previously reported that the methylation of L7 is inversely proportional to the growth temperature (Chang 1978), we now show that once L7 is methylated at 25 degree, the methyl group is stable when the culture is shifted to 37 degree C. However, a partial turnover of the methyl group of L7 is observed when the methylated ribosome is chased at 25 degree C. On the other hand, the methyl groups of L11 appear to be stable at either 25 degree C or 37 degree C. We also observe that the extent of methylation of both L7 and L11 stays nearly constant during the cell growth cycle from early log to stationary phase.

Influence of amino acid starvation on guanosine 5'-diphosphate 3'-diphosphate basal-level synthesis in Escherichia coli.

We observed that the synthesis of basal-level guanosine 5'-diphosphate 3'-diphosphate (ppGpp) in both relA mutants and relA+ relC strains of Escherichia coli decreased in response to amino acid limitation and that this was accompanied by an increase in ribonucleic acid (RNA) synthesis. Addition of the required amino acid to starved cultures of relaxed bacteria resulted in the resumption of ppGpp synthesis and a concomitant decrease in RNA production. Our results indicate that relA mutants retain a stringent factor-independent ribosomal mechanism for basal-level ppGpp synthesis. They also suggest that in relA+ bacteria, stringent factor-mediated ppGpp synthesis and the production of basal-level ppGpp are mutually exclusive. These findings substantiate the hypothesis that there are two functionally discrete mechanisms for ppGpp synthesis in E. coli. Through these studies we have also obtained new evidence which indicates that ppGpp serves as a modulator of RNA synthesis during balanced growth as well as under conditions of nutritional downshift and starvation.

Temperature-dependent variation in the extent of methylation of ribosomal proteins L7 and L12 in Escherichia coli.

The amount of epsilon-N-monomethyllysine in ribosomal proteins L7 and L12 in Escherichia coli is dependent upon the cell growth temperature. At 37 degrees C or above, very small amounts were detected. Dramatic increase in the content of epsilon-N-monomethyllysine in these proteins was observed when the growth temperature was lowered.

Identification of the methylated ribosomal proteins in HeLa cells and the fluctuation of methylation during the cell cycle.

Methylated proteins from HeLa cell cytoplasmic ribosomes have been identified. At least seven proteins are methylated and four of them are mildly acidic. The nature of the methylated amino acid in each protein is presented. In synchronized HeLa cell culture, the extent of methylation for both subunits varies with the cell cycle. Methylation of the 40 S subunit occurs heavily in the late G1 phase whereas methylation of the 60 S subunit is most pronounced in the early S phase.

Characterization of methylated neutral amino acids from Escherichia coli ribosomes.

The methylated neutral amino acids from both 30S and 50S ribosomal subunits of an Escherichia coli K strain were characterized. The 50S ribosomal subunit contains three methylated neutral amino acids: N-monomethylalanine, N-monomethylmethionine, and an as yet unidentified methylated amino acid found in protein L11. Both N-monomethylalanine and N-monomethylmethionine were found in protein L33. The amount of N-monomethylmethionine in this protein, however, is variable but not more than 0.25 molecules per protein. Thus protein L33 from this E. coli K strain has heterogeneity in its N-terminal amino acid and can start with either N-monomethylalanine or N-monomethylmethionine. The N-monomethylmethionine residue was not derived from the reduction of N-formylmethionine in the protein. The 30S ribosomal subunit contains only one methylated neutral amino acid: N-monomethylalanine.

Enzymatic methyl esterification of Escherichia coli ribosomal proteins.

Enzymatic methyl ester formation in Escherichia coli ribosomal proteins was observed. Alkali lability of the methylated proteins and derivatization of the methyl groups as methyl esters of 3,5-dinitrobenzoate indicate the presence of protein methyl esters. The esterification reaction occurs predominantly on the 30S ribosomal subunit, with protein S3 as the major esterified protein. When the purified 30S subunit was used as the methyl acceptor, protein S9 was also found to be esterified. The enzyme responsible for the esterification of free carboxyl groups in proteins, protein methylase II (S-adenosyl-L-methionine:protein carboxyl methyltransferase, EC 2.1.1.24), was identified in E. coli Q13. This enzyme is extremely unstable when compared with that from mammalian origin. By molecular sieve chromatography, E. coli protein methylase II showed multiple peaks, with a major broad peak around 120,000 daltons and several minor peaks in the lower-molecular-weight region. Rechromatography of the major enzyme peak showed activities in several fractions that are much lower in molecular weight. The substrate specificity of the E. coli enzyme is similar to that of the mammalian enzyme. The Km value for S-adenosyl-L-methionine is 1.96 X 10(-6) M, and S-adenosyl-L-homocysteine was found to be a competitive inhibitor, with a Ki value of 1.75 X 10(-6) M.