JoséL. García

Modular organization of the lytic enzymes of Streptococcus pneumoniae and its bacteriophages.

The nucleotide sequences of genes cpl7 and cpl9 of the Streptococcus pneumoniae bacteriophages Cp-7 and Cp-9, encoding the muramidases CPL-7 and CPL-9, respectively, have been determined. The N-terminal domains of CPL-7 and CPL-9 were virtually identical to that previously reported for the CPL-1 muramidase. The C-terminal domain of the CPL-7 muramidase, however, was different from those of the host amidase and the phage Cp-1 and Cp-9 lysozymes. Whereas all enzymes studied are characterized by repeated sequences at their C termini, the repeat-unit lengths are 20 amino acids (aa) in CPL-1, CPL-9 and in the host amidase, but 48 aa in CPL-7. Six repeated sequences represent the C-terminal domains of CPL-1, CPL-9 and the host amidase, and 2.8 perfect tandem repetitions that of CPL-7. The peculiar characteristics of the structure of CPL-7 muramidase correlate with its biochemical and biological properties. Whereas CPL-1, CPL-9 and the pneumococcal amidase strictly depend on the presence of choline-containing cell walls for activity, CPL-7 is able to degrade cell walls containing either choline or ethanolamine. These results support the previously postulated role for the C-terminal domain of these lytic enzymes in substrate recognition and provide further experimental evidence supporting the notion that the proteins have evolved by an exchange of modular units.

Nucleoid‐associated PhaF phasin drives intracellular location and segregation of polyhydroxyalkanoate granules in Pseudomonas putida KT2442

The PhaF is a nucleoid‐associated like protein of Pseudomonas putida KT2442 involved in the polyhydroxyalkanoate (PHA) metabolism. Its primary structure shows two modular domains; the N‐terminal PHA granule‐binding domain (phasin domain) and the C‐terminal half containing AAKP‐like tandem repeats characteristic of the histone H1 family. Although the PhaF binding to PHA granules and its role as transcriptional regulator have been previously demonstrated, the cell physiology meaning of these properties remains unknown. This work demonstrates that PhaF plays a crucial role in granule localization within the cell. TEM and flow cytometry studies of cells producing granules at early growth stage demonstrated that PhaF directs the PHA granules to the centre of the cells, forming a characteristic needle array. Our studies demonstrated the existence of two markedly different cell populations in the strain lacking PhaF protein, i.e. cells with and without PHA. Complementation studies definitively demonstrated a key role of PhaF in granule segregation during the cell division ensuring the equal distribution of granules between daughter cells. In vitro studies showed that PhaF binds DNA through its C‐terminal domain in a non‐specific manner. All these findings suggested a main role of PhaF in PHA apparatus through interactions with the segregating chromosome.

Structure of proline iminopeptidase from Xanthomonas campestris pv. citri: a prototype for the prolyl oligopeptidase family

The proline iminopeptidase from Xanthomonas campestris pv. citri is a serine peptidase that catalyses the removal of N‐terminal proline residues from peptides with high specificity. We have solved its three‐dimensional structure by multiple isomorphous replacement and refined it to a crystallographic R‐factor of 19.2% using X‐ray data to 2.7 Å resolution. The protein is folded into two contiguous domains. The larger domain shows the general topology of the α/β hydrolase fold, with a central eight‐stranded β‐sheet flanked by two helices and the 11 N‐terminal residues on one side, and by four helices on the other side. The smaller domain is placed on top of the larger domain and essentially consists of six helices. The active site, located at the end of a deep pocket at the interface between both domains, includes a catalytic triad of Ser110, Asp266 and His294. Cys269, located at the bottom of the active site very close to the catalytic triad, presumably accounts for the inhibition by thiol‐specific reagents. The overall topology of this iminopeptidase is very similar to that of yeast serine carboxypeptidase. The striking secondary structure similarity to human lymphocytic prolyl oligopeptidase and dipeptidyl peptidase IV makes this proline iminopeptidase structure a suitable model for the three‐dimensional structure of other peptidases of this family.

Interchange of functional domains switches enzyme specificity: construction of a chimeric pneumococcal‐clostridial cell wall lytic enzyme

Bacterial autolysins are endogenous enzymes that specifically cleave covalent bonds in the cell wall. These enzymes show both substrate and bond specificities. The former is related to their interaction with the insoluble substrate whereas the latter determine their site of action. The bond specificity allows their classification as muramidases (lysozymes), glucosaminldases, amidases, and endopeptidases. To demonstrate that the autolysin (LYC muramidase) of Clostridium acetobutylicum ATCC824 presents a domainal organization, a chimeric gene (clc) containing the regions coding for the catalytic domain of the LYC muramidase and the choline‐binding domain of the pneumococcal phage CPL1 muramidase has been constructed by in vitro recombination of the corresponding gene fragments. This chimeric construction codes for a choline‐binding protein (CLC) that has been purified using affinity chromatography on DEAE‐cellulose. Several biochemical tests demonstrate that this rearrangement of domains has generated an enzyme with a choline‐dependent muramidase activity on pneumococcal cell walls. Since the parental LYC muramidase was cholineindependent and unable to degrade pneumococcal cell walls, the formation of this active chimeric enzyme by exchanging protein domains between two enzymes that specifically hydrolyse cell walls of bacteria belonging to different genera shows that a switch on substrate specificity has been achieved. The chimeric CLC muramidase behaved as an autolytic enzyme when it was adsorbed onto a live autolysin‐defective mutant of Streptococcus pneumoniae. The construction described here provides experimental support for the theory of modular evolution which assumes that novel proteins have evolved by the assembly of preexisting polypeptide units.

Studies on the structure and function of the N-terminal domain of the pneumococcal murein hydrolases

The structures of the choline‐dependent pneumococcal murein hydrolases, LYTA amtdase and CPL1 Iysozyme, and the choline‐independent CPL7 Iysozyme were analysed by controlled proteolytic digestions. The trypsin cleavage of the CPL1 and CPL7 lysozymes produced two resistant polypeptides, F1 and F7 respectively, corresponding to the N‐terminal domain of the enzymes, whereas the amidase LYTA was completely hydrolysed by the protease. Interestingly, the F1 and F7 fragments showed a low, but significant, choline‐independent lysozyme activity. Choline reduced the rate of proteolytic hydrolysis of choline‐dependent enzymes, suggesting that the C‐terminal choline‐binding domain adopts a more resistant conformation in the presence of the ligand. On the other hand, the regions encoding the N‐terminal domains of the three enzymes have been cloned and expressed in Escherichia coli, showing that these domains adopt an active conformation even in the absence of their C‐terminal domains. The lower activity shown by the catalytic domains when compared with that of the complete enzymes suggests that the acquisition of a substrate‐binding domain represents a noticeable evolutionary advantage for enzymes that interact with polymeric substrates, allowing them to achieve a higher catalytic efficiency. These results strongly reinforce the hypothesis that the pneumococcal murein hydrolases have been originated by fusion of two structural and functional independent domains, and provide new experimental support to the theory of modular evolution of proteins.

Cloning and sequencing of the pac gene encoding the penicillin G acylase of Bacillus megaterium ATCC 14945

The pac gene encoding the penicillin G acylase (PGA) of Bacillus megaterium ATCC 14945 has been cloned in Escherichia coli HB101 (proA, leuB) using a selective minimal medium containing phenylacetyl-L-leucine instead of L-leucine. The nucleotide sequence of this gene has been determined and contains an open reading frame of 2406 nucleotides. The deduced amino acid sequence shows significant similarity with other beta-lactam acylases. Although the PGA of B. megaterium is extracellular, the enzyme produced in E. coli appears to have a cytoplasmic localization.

Sequence of the lye gene encoding the autolytic lysozyme of Clostridium acetobutylicum ATCC824: comparison with other lytic enzymes

The lyc gene, encoding an autolytic lysozyme from Clostridium acetobutylicum ATCC824, has been cloned. The nucleotide sequence of the lyc gene has been determined and found to encode a protein of 324 amino acids (aa) with a deduced Mr of 34,939. The lyc gene is preceded by two open reading frames with unknown functions, suggesting that this gene is part of an operon. Comparison between the deduced aa sequence of the lyc gene and the directly determined N-terminal sequence of the extracellular clostridial lysozyme suggests that the enzyme is synthesized without a cleavable signal peptide. Moreover, the comparative analyses between the clostridial lysozyme and other known cell-wall lytic enzymes revealed a significant similarity with the N-terminal portion of the lysozymes of Streptomyces globisporus, the fungus Chalaropsis, the Lactobacillus bulgaricus bacteriophage mv1, and the Streptococcus pneumoniae bacteriophages of the Cp family (CPL lysozymes). In addition, the analyses showed that the C-terminal half of the clostridial lysozyme was homologous to the N-terminal domain of the muramoyl-pentapeptide-carboxypeptidase of Streptomyces albus, suggesting a role in substrate binding. The existence of five putative repeated motifs in the C-terminal region of the autolytic lysozyme suggests that this region could play a role in the recognition of the polymeric substrate.

Identification of the 4-hydroxyphenylacetate transport gene of Escherichia coli W : construction of a highly sensitive cellular biosensor

The mechanism of uptake of 4‐hydroxyphenylacetate (4‐HPA) by Escherichia coli W. was investigated. The 4‐HPA uptake was induced by 4‐HPA, 3‐hydroxyphenylacetate (3‐HPA) or phenylacetate (PA) and showed saturation kinetics with apparent K t and V max values of 25 μM and 3 nmol/min per 109 cells, respectively. Transport of 4‐HPA was resistant to N,N″‐dimethylcarbodiimide (DCCD), but was completely inhibited by cyanide and 4‐nitrophenol, and, to a lower extent, by arsenate and azide, suggesting that energy is required for the uptake process. Competition studies showed that 4‐HPA uptake was inhibited by 3‐HPA or 3,4‐dihydroxyphenylacetate (3,4‐DHPA) but not by 2‐hydroxyphenylacetate (2‐HPA), l‐tyrosine or other structural analogues, indicating a narrow specificity of the transport system. We have demonstrated, using two experimental approaches, that the hpaX gene of the 4‐HPA catabolic cluster, which encodes a protein of the superfamily of transmembrane facilitators, is responsible for 4‐HPA transport. Aside from the aromatic amino acid transport systems, hpaX is the first transport gene for an aromatic compound of enteric bacteria that has been characterized. A highly sensitive cellular biosensor has been constructed by coupling the 4‐HPA transport system to a regulatory circuit that controls the production of β‐galactosidase. This biosensor has allowed us to demonstrate that the transport system performs efficiently at very low external concentrations of 4‐HPA, similar to levels that would be expected to occur in natural environments.

Construction of a broad-host-range pneumococcal promoter-probe plasmid

A promoter-probe plasmid, pLSE4, containing the promoterless lytA gene that encodes the major pneumococcal autolysin, was developed to isolate and characterize nucleotide sequences of Streptococcus pneumoniae involved in transcriptional regulation. This vector was derived from the broad-host-range plasmid pLS1 and is suitable for the transformation of Gram- and Gram+ bacteria. An array of unique restriction sites was placed upstream from the lytA coding region. Pneumococcal promoters can be screened from random DNA fragments cloned in these sites for the ability to direct the expression of the autolysin in transformed autolysin-deficient pneumococcal cells. Transformants showing a Lyt+ phenotype were selected on agar plates using a simple filter technique. Relative promoter strength was determined by direct assay of the cell wall lytic activity in cell extracts.

Molecular characterization of an autolysin-defective mutant of Streptococcuspneumoniae

The mutant gene lyt-4 of the autolysin-defective mutant R6ly4-4 of Streptococcus pneumoniae, which synthesized a temperature-sensitive autolytic enzyme, has been cloned in Escherichia coli. The nucleotide defect of the lyt-4 mutation has been characterized as a CG to TA transition. This transition causes the appearance of a glutamic acid instead of a glycine in the amino acid sequence of the autolysin, altering the hydropathic profile of the protein. This alteration might explain the observed thermosensitivity of the mutated autolytic enzyme. The present work represents the first molecular characterization of a mutation in the structural gene of a bacterial autolysin.