Protein phosphorylation and regulation of carbon metabolism in gram-negative versus gram-positive bacteria.

Bacteria impose regulatory mechanisms on metabolic processes to ensure that the needs of the cell are met but not exceeded. Here, we discuss the basic features of a mechanism by which carbohydrate catabolism in Gram-positive bacteria is regulated. Although the physiological consequences of this regulation are the same as in Gram-negative bacteria, the mechanism is entirely different. These regulatory processes evidently evolved late, after the divergence of Gram-negative bacteria, even though the targets of regulation are universal.

Distinct affinity of binding sites for S-layer homologous domains in Clostridium thermocellum and Bacillus anthracis cell envelopes.

Binding parameters were determined for the SLH (S-layer homologous) domains from the Clostridium thermocellum outer layer protein OlpB, from the C. thermocellum S-layer protein SlpA, and from the Bacillus anthracis S-layer proteins EA1 and Sap, using cell walls from C. thermocellum and B. anthracis. Each SLH domain bound to C. thermocellum and B. anthracis cell walls with a different KD, ranging between 7.1 x 10(-7) and 1.8 x 10(-8) M. Cell wall binding sites for SLH domains displayed different binding specificities in C. thermocellum and B. anthracis. SLH-binding sites were not detected in cell walls of Bacillus subtilis. Cell walls of C. thermocellum lost their affinity for SLH domains after treatment with 48% hydrofluoric acid but not after treatment with formamide or dilute acid. A soluble component, extracted from C. thermocellum cells by sodium dodecyl sulfate treatment, bound the SLH domains from C. thermocellum but not those from B. anthracis proteins. A corresponding component was not found in B. anthracis.

Fluorometric assay for the measurement of viral neuraminidase in influenza vaccines.

Influenza viruses have two surface glycoproteins: haemagglutinin and neuraminidase which are capable of inducing a significant antibody response following vaccination. All neuraminidases from different strains of influenza A and B viruses are able to hydrolyse alpha-ketosidic linkages between N-acetylneuraminic (sialic) acid and other carbohydrates. In this report, the neuraminidase activity was assayed in various influenza vaccines by using a fluorogenic substrate: the sodium salt of 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid. This method was reliable (variation less than 8%) and more sensitive (100 to 1000 times) in less time (incubation time = 15 min) than the Warren assay. Therefore, the method is suitable for the control of the sialidase activity during the processing of influenza vaccines.

Site-directed mutagenesis of essential carboxylic residues in Clostridium thermocellum endoglucanase CelD.

12 carboxyl residues of the Clostridium thermocellum endoglucanase CelD were mutated to alanine. The specific activity of five of the mutated proteins was 1% or less that of wild type. The Ca2+ binding isotherms of these five were similar to those of wild type CelD, consistent with the fact that none of the mutated residues is observed to be directly involved in Ca2+ binding in the three-dimensional structure of the protein (Juy, M., Anit, A. G., Alzuri, P. M., Poljak, R. J., Claeyssens, M., Béguin, P., and Aubert, J.-P., manuscript in preparation) and suggesting that the mutations did not result in gross alterations of the tertiary structure. Analysis of the physico-chemical and enzymatic properties of the five purified mutated proteins and consideration of their position in the three-dimensional structure suggest that carboxyl groups identified may play roles as a general acid catalyst and a source of negative charge in stabilizing a carbonium ion intermediate. Among mutated residues, Glu-555 appears as the most likely candidate for participating in the catalytic mechanism of endoglucanase CelD.

CcpB, a novel transcription factor implicated in catabolite repression in Bacillus subtilis.

Recent work has shown that in Bacillus subtilis catabolite repression of several operons is mediated by a mechanism dependent on DNA-binding protein CcpA complexed to a seryl-phosphorylated derivative of HPr [HPr(Ser-P)], the small phosphocarrier protein of the phosphoenolpyruvate-sugar phosphotransferase system. In this study, it was found that a transposon insertional mutation resulted in the partial loss of gluconate (gnt) and xylose (xyl) operon catabolite repression by glucose, mannitol, and sucrose. The transposon insertion was localized to a gene, designated ccpB, encoding a protein 30% identical to CcpA, and relief from catabolite repression was shown to be due to the absence of CcpB rather than to the absence of a protein encoded by a downstream gene within the same operon. The relative intensities of CcpA- and CcpB-mediated catabolite repression depended on growth conditions. On solid media, and when cells were grown in liquid media with little agitation, CcpB and CcpA both proved to function in catabolite repression. However, when cells were grown in liquid media with much agitation, CcpA alone mediated catabolite repression. Like CcpA, CcpB appears to exert its catabolite-repressing effect by a mechanism dependent on the presence of HPr(Ser-P).

Identification of a histidyl residue in the active center of endoglucanase D from Clostridium thermocellum.

Diethylpyrocarbonate modification of endoglucanase D from Clostridium thermocellum, cloned in Escherichia coli, resulted in a rapid but partial (maximally 70-80%) loss of activity. The second-order rate constant of inactivation proved to be exceptionally high (3210 M-1.min-1). A 3-fold reduction of the kcat and a 2-fold increase of the Km for 2'-chloro-4'-nitrophenyl beta-cellobioside were observed. Spectrophotometric analysis indicate the presence of one rapidly (k = 0.45 min-1) and two slower (k = 0.23 min-1) reacting histidyl residues. In the presence of 50 mM methyl beta-cellotrioside, the rate of inactivation was reduced 16-fold, and the kinetics of modification were compatible with the protection of 1 histidyl residue. Since peptide analysis was inconclusive, identification of the critical residue was attempted by site-directed mutagenesis. Each of the 12 histidyl residues present in the endoglucanase D sequence was mutated into either Ala or Ser. Seven of the mutant enzymes had specific activities lower than 50% of the wild-type. Only in the case of the Ser-516 mutant, however, was the residual activity not affected by diethyl pyrocarbonate. These findings suggest an important functional or structural role for His-516 in the wild-type enzyme.

Characterization of glucose-specific catabolite repression-resistant mutants of Bacillus subtilis: identification of a novel hexose:H+ symporter.

Insertional mutagenesis was conducted on Bacillus subtilis cells to screen for mutants resistant to catabolite repression. Three classes of mutants that were resistant to glucose-promoted but not mannitol-promoted catabolite repression were identified. Cloning and sequencing of the mutated genes revealed that the mutations occurred in the structural genes for (i) enzyme II of the phosphoenolpyruvate-glucose phosphotransferase (PtsG), (ii) antiterminator GlcT, which controls PtsG synthesis, and (iii) a previously uncharacterized carrier of the major facilitator superfamily, which we have designated GlcP. The last protein exhibits greatest sequence similarity to the fucose:H+ symporter of Escherichia coli and the glucose/galactose:H+ symporter of Brucella abortus. In a wild-type B. subtilis genetic background, the glcP::Tn10 mutation (i) partially but specifically relieved glucose- and sucrose-promoted catabolite repression, (ii) reduced the growth rate in minimal glucose medium, and (iii) reduced rates of [14C]glucose and [14C]methyl alpha-glucoside uptake. In a delta pts genetic background no phenotype was observed, suggesting that expression of the glcP gene required a functional phosphotransferase system. When overproduced in a delta pts mutant of E. coli, GlcP could be shown to specifically transport glucose, mannose, 2-deoxyglucose and methyl alpha-glucoside with low micromolar affinities. Accumulation of the nonmetabolizable glucose analogs was demonstrated, and inhibitor studies suggested a dependency on the proton motive force. We conclude that B. subtilis possesses at least two distinct routes of glucose entry, both of which contribute to the phenomenon of catabolite repression.

Structural and functional analysis of the metal-binding sites of Clostridium thermocellum endoglucanase CelD.

Crystallographic analysis indicated that Clostridium thermocellum endoglucanase CelD contained three Ca(2+)-binding sites, termed A, B, and C, and one Zn(2+)-binding site. The protein contributed five, six, and three of the coordinating oxygen atoms present at sites A, B, and C, respectively. Proteins altered by mutation in site A (CelDD246A), B (CelDD361A), or C (CelDD523A) were compared with wild type CelD. The Ca(2+)-binding isotherm of wild type CelD was compatible with two high affinity sites (Ka = 2 x 10(6) M-1) and one low affinity site (Ka < 10(5) M-1). The Ca(2+)-binding isotherms of the mutated proteins showed that sites A and B were the two high affinity sites and that site C was the low affinity site. Atomic absorption spectrometry confirmed the presence of one tightly bound Zn2+ atom per CelD molecule. The inactivation rate of CelD at 75 degrees C was decreased 1.9-fold upon increasing the Ca2+ concentration from 2 x 10(-5) to 10(-3) M. The Km of CelD was decreased 1.8-fold upon increasing the Ca2+ concentration from 5 x 10(-6) to 10(-4) M. Over similar ranges of concentration, Ca2+ did not affect the thermostability nor the kinetic properties of CelDD523A. These findings suggest that Ca2+ binding to site C stabilizes the active conformation of CelD in agreement with the close vicinity of site C to the catalytic center.

Calcium-binding affinity and calcium-enhanced activity of Clostridium thermocellum endoglucanase D.

Clostridium thermocellum endoglucanase D (EC 3.2.1.4: EGD), which is encoded by the celD gene, was found to bind Ca2+ with an association constant of 2.03 x 10(6) M-1. Ca2+ stimulated the activity of EGD towards swollen Avicel by 2-fold. In the presence of Ca2+, the Kd of the enzyme towards p-nitrophenyl-beta-D-cellobioside and carboxymethylcellulose was decreased by 4-fold. Furthermore, Ca2+ increased the half-life of the enzyme at 75 degrees C from 13 to 47 min. Since the 3' sequence of celD encodes a duplicated region sharing similarities with the Ca2+-binding site of several Ca2+-binding proteins, a deleted clone was constructed and used to purify a truncated form of the enzyme which no longer contained the duplicated region. The truncated enzyme was very similar to EGD expressed from the intact gene with respect to activity, Ca2(+)-binding kinetics and Ca2+ effects on substrate binding and thermostability. Thus the latter parameters do not appear to be mediated through the duplicated conserved region.

Cloning of a genetically unstable cytochrome P-450 gene cluster involved in degradation of the pollutant ethyl tert-butyl ether by Rhodococcus ruber.

Rhodococcus ruber (formerly Gordonia terrae) IFP 2001 is one of a few bacterial strains able to degrade ethyl tert-butyl ether (ETBE), which is a major pollutant from gasoline. This strain was found to undergo a spontaneous 14.3-kbp chromosomal deletion, which results in the loss of the ability to degrade ETBE. Sequence analysis of the region corresponding to the deletion revealed the presence of a gene cluster, ethABCD, encoding a ferredoxin reductase, a cytochrome P-450, a ferredoxin, and a 10-kDa protein of unknown function, respectively. The EthB and EthD proteins could be easily detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were induced by ETBE in the wild-type strain. Upstream of ethABCD lies ethR, which codes for a putative positive transcriptional regulator of the AraC/XylS family. Transformation of the ETBE-negative mutant by a plasmid carrying the ethRABCD genes restored the ability to degrade ETBE. Complementation was abolished if the plasmid carried ethRABC only. The eth genes are located in a DNA fragment flanked by two identical direct repeats of 5.6 kbp. The ETBE-negative mutants carry a single copy of this 5.6-kbp repeat, suggesting that the 14.3-kbp chromosomal deletion resulted from a recombination between the two identical sequences. The 5.6-kbp repeat is a class II transposon carrying a TnpA transposase, a truncated form of the recombinase TnpR, and a terminal inverted repeat of 38 bp. The truncated TnpR is encoded by an IS3-interrupted tnpR gene.

A common protein fold and similar active site in two distinct families of beta-glycanases.

The structure of Clostridium thermocellum endoglucanase CelC, a member of the largest cellulase family (family A), has been determined at 2.15 A resolution. The protein folds into an (alpha/beta)8 barrel, with a deep active-site cleft generated by the insertion of a helical subdomain. The structure of the catalytic core of xylanase XynZ, which belongs to xylanase family F, has been determined at 1.4 A resolution. In spite of significant differences in substrate specificity and structure (including the absence of the helical subdomain), the general polypeptide folding pattern, architecture of the active site and catalytic mechanism of XynZ and CelC are similar, suggesting a common evolutionary origin.