A major autolysin of Pseudomonas aeruginosa: subcellular distribution, potential role in cell growth and division and secretion in surface membrane vesicles.

A 26-kDa murein hydrolase is the major autolysin of Pseudomonas aeruginosa PAO1, and its expression can be correlated with the growth and division of cells in both batch and synchronously growing cultures. In batch cultures, it is detected primarily during the mid-exponential growth phase, and in synchronous cultures, it is detected primarily during the cell elongation and division phases. Immunogold labeling of thin sections of P. aeruginosa using antibodies raised against the 26-kDa autolysin revealed that it is associated mainly with the cell envelope and in particular within the periplasm. It is also tightly bound to the peptidoglycan layer, since murein sacculi, isolated by boiling 4% sodium dodecyl sulfate treatment, could also be immunogold labeled. Since division is due to cell constriction in this P. aeruginosa strain (septa are rarely seen), we cannot comment on the autolysin's contribution to septation, although constriction sites were always heavily labeled. Some labeling was also found in the cytoplasm, and this was thought to be due to the de novo synthesis of the enzyme before translocation to the periplasm. Interestingly, the autolysin was also found to be associated with natural membrane vesicles which blebbed from the surface during cell growth; the enzyme is therefore part of the complex makeup of these membrane packages of secreted materials (J. L. Kadurugamuwa and T. J. Beveridge, J. Bacteriol. 177:3998-4008, 1995). The expression of these membrane vesicles was correlated with the expression of B-band lipopolysaccharide.     

Bacteriolytic effect of membrane vesicles from Pseudomonas aeruginosa on other bacteria including pathogens: conceptually new antibiotics.

Pseudomonas aeruginosa releases membrane vesicles (MVs) filled with periplasmic components during normal growth, and the quantity of these vesicles can be increased by brief exposure to gentamicin. Natural and gentamicin-induced membrane vesicles (n-MVs and g-MVs, respectively) are subtly different from one another, but both contain several important virulence factors, including hydrolytic enzyme factors (J. L. Kadurugamuwa and T. J. Beveridge, J. Bacteriol. 177:3998-4008, 1995). Peptidoglycan hydrolases (autolysins) were detected in both MV types, especially a periplasmic 26-kDa autolysin whose expression has been related to growth phase (Z. Li, A. J. Clarke, and T. J. Beveridge, J. Bacteriol. 178:2479-2488, 1996). g-MVs possessed slightly higher autolysin activity and, at the same time, small quantities of gentamicin. Both MV types hydrolyzed isolated gram-positive and gram-negative murein sacculi and were also capable of hydrolyzing several glycyl peptides. Because the MVs were bilayered, they readily fused with the outer membrane of gram-negative bacteria. They also adhered to the cell wall of gram-positive bacteria. g-MVs were more effective in lysing other bacteria because, in addition to the autolysins, they also contained small amounts of gentamicin. The bactericidal activity was 2.5 times the MIC of gentamicin, which demonstrates the synergistic effect of the antibiotic with the autolysins. n-MVs were capable of killing cultures of P. aeruginosa with permeability resistance against gentamicin, indicating that the fusion of n-MV to the outer membrane liberated autolysins into the periplasm, where they degraded the peptidoglycan and lysed the cells. g-MVs had even greater killing power since they liberated both gentamicin and autolysins into these resistant cells. These findings may help develop a conceptually new group of antibiotics designed to be effective against hard-to-kill bacteria.     

Analysis of the sodium dodecyl sulfate-stable peptidoglycan autolysins of select gram-negative pathogens by using renaturing polyacrylamide gel electrophoresis.

For the first time, peptidoglycan autolysins from cellular fractions derived from sonicated cultures of Pseudomonas aeruginosa PAO1, Escherichia coli W7, Klebsiella pneumoniae CWK2, and Proteus mirabilis 19 were detected and partially characterized by zymogram analysis. Purified murein sacculi from P. aeruginosa PAO1 were incorporated into a sodium dodecyl sulfate (SDS)-polyacrylamide gel at a concentration of 0.05% (wt/vol) to serve as a substrate for the separated autolysins. At least 11 autolysin bands of various intensities with M(r)s ranging between 17,000 and 122,000 were detected in each of the homogenated cultures. Some of the autolysins of the four bacteria had similar M(r)s. The zymogram analysis was used to show that a number of the autolysins from E. coli were inhibited by the heavy metals Hg2+ and Cu2+, at 1 and 10 mM, respectively, high ionic strengths, and reagents known to affect the packing of lipopolysaccharides. The activity of an autolysin with an M(r) of 65,000 was also impaired by penicillin G, whereas it was enhanced by gentamicin. A preliminary screen to determine the relationship between penicillin-binding proteins (PBPs) and autolysins was carried out by using a dual assay in which radiolabelled penicillin V bands were visualized on an autolysin zymogram. Radiolabelled bands corresponding to PBPs 3, 4, 5, and 6 from E. coli and P. aeruginosa; PBPs 3, 4, and 6 from Proteus mirabilis; and PBP 6 from K. pneumoniae degraded the murein sacculi in the gels and were presumed to have autolytic activity, although the possibility of two distinct enzymes, each with one of the activities, comigrating in the SDS-polyacrylamide gels could not be excluded. Some radiolabelled bands possessed an Mr of <34,000 and coincided with similar low-Mr autolysin bands.     

Isolation and purification of a Campylobacter upsaliensis autolysin.

Autolytic activity in the soluble and sediment fractions of sonicates of the spiral and the coccoid form of Campylobacter upsaliensis could not be demonstrated by native (nondenaturing) polyacrylamide gel electrophoresis (PAGE). Autolysins were detected, however, by using denaturing sodium dodecyl sulfate (SDS)-PAGE gels containing either purified Escherichia coli peptidoglycan or whole cells of Micrococcus luteus (Micrococcus lysodeikticus) as the turbid substrate, with subsequent renaturation by treatment with Triton X-100 buffer. In renaturing gels that contained Escherichia coli peptidoglycan, 14 putative autolytic bands ranging from 200 to 12 kDa were detected. In similar gels containing whole cells of M. luteus, only a single band appeared with a molecular mass of 34 kDa. This band corresponded to one of the bands present in the gels containing Escherichia coli peptidoglycan. This common autolysin was isolated by adsorbing it from Campylobacter upsaliensis soluble fractions onto M. luteus cells and then subjecting these cells to renaturing SDS-PAGE in gels containing Escherichia coli peptidoglycan. The 34-kDa autolysin differed from a single 51-kDa autolysin unique to the M. luteus cells, and when isolated from an SDS-PAGE gel, was pure when tested by isoelectric focusing. The N-terminal amino acid sequence analysis showed the first 15 amino acids of the 34-kDa autolysin to have 67% identity to a part of antigenic protein PEB4 of Campylobacter jejuni. The purified autolysin was used to immunize rabbits and the antibodies produced precipitated autolytic activity from cell lysates. The specificity of the antibodies was shown by Western blotting: only a single specific band occurred, with a molecular mass of 34 kDa, and thus it seems unlikely that the 34-kDa autolysin was derived from any of the other autolysins that were detected.     

A novel lipolytic enzyme located in the outer membrane of Pseudomonas aeruginosa

A lipase-negative deletion mutant of Pseudomonas aeruginosa PAO1 still showed extracellular lipolytic activity toward short-chain p-nitrophenylesters. By screening a genomic DNA library of P. aeruginosa PAO1, an esterase gene, estA, was identified, cloned, and sequenced, revealing an open reading frame of 1,941 bp. The product of estA is a 69.5-kDa protein, which is probably processed by removal of an N-terminal signal peptide to yield a 67-kDa mature protein. A molecular mass of 66 kDa was determined for (35)S-labeled EstA by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The amino acid sequence of EstA indicated that the esterase is a member of a novel GDSL family of lipolytic enzymes. The estA gene showed high similarity to an open reading frame of unknown function located in the trpE-trpG region of P. putida and to a gene encoding an outer membrane esterase of Salmonella typhimurium. Amino acid sequence alignments led us to predict that this esterase is an autotransporter protein which possesses a carboxy-terminal beta-barrel domain, allowing the secretion of the amino-terminal passenger domain harboring the catalytic activity. Expression of estA in P. aeruginosa and Escherichia coli and subsequent cell fractionation revealed that the enzyme was associated with the cellular membranes. Trypsin treatment of whole cells released a significant amount of esterase, indicating that the enzyme was located in the outer membrane with the catalytic domain exposed to the surface. To our knowledge, this esterase is unique in that it exemplifies in P. aeruginosa (i) the first enzyme identified in the outer membrane and (ii) the first example of a type IV secretion mechanism.     

Bactericidal effect of gentamicin-induced membrane vesicles derived from Pseudomonas aeruginosa PAO1 on gram-positive bacteria

Previous studies have shown that gentamicin-induced membrane vesicles (g-MVs) from Pseudomonas aeruginosa PAO1 possess both the antibiotic (gentamicin) and a potent peptidoglycan hydrolase (PGase; autolysin) that is effective in killing gram-negative pathogens. This present study evaluated the therapeutic potential of g-MVs against four gram-positive bacteria. Bactericidal assays and electron microscopy of thin sections revealed that Bacillus subtilis 168 and Staphylococcus aureus D2C were susceptible to killing mediated by g-MVs, Listeria monocytogenes ATCC 19113 was slightly susceptible, whereas Enterococcus hirae ATCC 9790 was unaffected. g-MVs were generally more effective against the bacteria than was soluble gentamicin, suggesting they could have more killing power than natural membrane vesicles containing no antibiotic. Electron microscopy and hydrophobic interaction chromatography showed that more membrane vesicles (MVs) initially attached to B. subtilis (hydrophilic) than to predominantly hydrophobic E. hirae, L. monocytogenes, and S. aureus. Zymograms containing murein sacculi as an enzyme substrate illustrated that all organisms except E. hirae were sensitive to the 26-kDa autolysin to varying degrees. Peptidoglycan O-acetylation did not influence susceptibility to MV-mediated lysis. Though not universally effective, the g-MV delivery system remains a promising therapeutic alternative for specific gram-positive infections.     

A peptidoglycan hydrolase similar to bacteriophage endolysins acts as an autolysin in Neisseria gonorrhoeae

We have identified a gene encoding an autolysin (atlA) from Neisseria gonorrhoeae . The deduced amino acid sequence of AtlA shows significant similarity to the peptidoglycan degrading transglycosylases (endolysins) of bacteriophages lambda and P2, suggesting that the encoded protein also functions in peptidoglycan hydrolysis. An atlA mutant was identical to the wild-type strain in exponential growth rate, but demonstrated reduced lysis and peptidoglycan turnover in the stationary phase of growth. When transferred into a buffer solution, at a pH non-permissive for other gonococcal autolysins, an autolytic activity was detectable in the wild-type strain that was not present in the mutant. The most dramatic phenotype of the mutant occurred after extended time in stationary phase. After approximately 16 h in stationary phase, both strains underwent an apparent replication event, after which the wild-type strain died rapidly whereas the atlA mutant survived considerably longer. Even after both the wild-type and mutant cells were dead, many of the mutant cells maintained intact morphology, whereas the wild-type cells were lysed. These results suggest that AtlA is a peptidoglycan transglycosylase related to bacteriophage endolysins and acts as an autolysin in the stationary phase.     

Analysis of the autolysins of Bacillus subtilis 168 during vegetative growth and differentiation by using renaturing polyacrylamide gel electrophoresis.

The autolysins of Bacillus subtilis 168 were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with substrate-containing gels. Four bands of vegetative autolytic activity of 90, 50, 34, and 30 kDa (bands A1 to A4) were detected in SDS and LiCl extracts and in native cell walls by using B. subtilis 168 vegetative cell walls as the substrate incorporated in the gel. The four enzyme activities showed different substrate specificities and sensitivities to various chemical treatments. The autolysin profile was not medium dependent and remained constant during vegetative growth. During sporulation, band A4 greatly increased in activity just prior to mother-cell lysis. No germination-associated changes in the profile were observed, although a soluble 41-kDa endospore-associated cortex-lytic enzyme was found. By using insertionally inactivated mutants, bands A1 and A2 were positively identified as the previously characterized 90-kDa glucosaminidase and 50-kDa amidase, respectively. The common filamentous phenotype of various regulatory mutants could not be correlated to specific changes in the autolysin profile.     

Zymogram and Preliminary Characterization of Lactobacillus helveticus Autolysins

The autolysins of Lactobacillus helveticus ISLC5 were detected and partially characterized by renaturing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with substrate-containing gels (zymogram). By using lyophilized Micrococcus luteus cells or heated whole cells of L. helveticus ISLC5 (0.2% [wt/vol]) as a substrate, several lytic activities were detected in the whole-cell SDS extract of strain ISLC5 (i) one activity at 42.4 kDa, which was named autolysin A, and (ii) six other activities having very similar molecular weights (29.1, 29.6, 30, 30.8, 31.7, and 32.8 kDa), which were named autolysins B (B1 through B6, respectively). As regards the temporal distribution of the enzymes, autolysins A and B were detected in the cells harvested from the beginning of the exponential growth phase. Autolysin A appeared to be associated only with viable cells, whereas the autolysins B remained associated with the cell envelope several days after the complete loss of culture viability. When SDS-treated walls of L. helveticus ISLC5 were used as a substrate, a supplementary lytic activity appeared at 37.5 kDa; it was considered a peptidoglycan hydrolase, since it was not able to induce lysis of whole-cell substrate. The autolysins of 30 other strains of L. helveticus from various geographical origins were also analyzed by zymogram; all the activity profiles obtained were similar to that of strain ISLC5 in terms of the number of lytic bands and their apparent molecular weights. Only the relative intensities of the lytic bands corresponding to autolysins A and B were variable depending on the strains. This observation suggested that autolysins are highly conserved enzymes. A concentrated crude lysate of the virulent bacteriophage 832-B1 infecting L. helveticus was also analyzed by zymogram; one lytic activity with an apparent molecular weight of 31.7 kDa, very close to the weights of the autolysins B, was observed. Finally, the autolysins of L. helveticus ISLC5 were successfully extracted from whole cells by using a 1 M lithium chloride solution; they were partially purified by precipitation, selective resolubilization, and gel filtration chromatography, which led to a 20-fold increase in specific activity.     

Enhanced butanol production and reduced autolysin activity after chloramphenicol treatment of Clostridium acetobutylicum ATCC 824

Summary Release of autolysin during the late exponential growth phase of Clostridium acetobutylicum resulted in early lysis of the culture and reduction of solvent formation. A simple and effective way of reducing autolysin activity and increasing solvent production is partial inhibition of protein synthesis with chloramphenicol (CAP). The extracellular autolytic activity in the culture, determined by following loss of turbidity of washed clostridial cells in 0.04m sodium phosphate buffer at 37° C, was decreased by 40% after CAP treatment. This caused an extension of cell viability by 12 h and an increase in butanol production by 30%. The optimal time of CAP addition was 12 h of incubation, and the optimal antibiotic concentration was 120 g/ml. The effects of CAP on the fermentation are due to the inhibition of protein synthesis leading to a decrease in autolysin level in the culture. The results obtained provide economic advantages for industrial production of solvents by minimizing autolysin activity and maximizing solvent yield during the critical solvent-producing phase. Correspondence to: R. W. Traxler     

Efficient production and processing of elastase and LasA by Pseudomonas aeruginosa require zinc and calcium ions.

The ability of Pseudomonas aeruginosa to degrade elastin, a major component of connective tissue, likely contributes to its pathogenicity and multiplication in human tissues. Two extracellular enzymes are required for P. aeruginosa elastolytic activity: elastase and LasA. Elastase is a zinc metalloprotease, but little is known about the structure of LasA. When grown under metal ion-deficient conditions, P. aeruginosa culture supernatants were found to exhibit a low level of elastolytic activity, which coincided with production of low levels of the 51-kDa proelastase and no detectable LasA. By using this fact to identify factors that promote elastolytic activity, P. aeruginosa PAO1, FRD2, and DG1 were grown in metal ion-deficient medium supplemented with zinc (10(-4) M ZnCl2), calcium (2.5 x 10(-3) M CaCl2), or iron (10(-4) M FeCl3). High levels of proteolytic and elastolytic activity were exhibited by all strains when cultured in the presence of both zinc and calcium, and this was associated with the production of mature 33-kDa elastase and 21-kDa LasA. Supplementing DG1 and PAO1 cultures with zinc alone stimulated the production of 33-kDa elastase, which, because of the calcium-deficient conditions, exhibited low proteolytic and elastolytic activities. Zinc also stimulated the production of a 41-kDa form of LasA in DG1 and PAO1 culture supernatants. Elastase production by FRD2 cultured in the presence of zinc alone differed from that by the other two strains in that supernatants contained 33-kDa elastase, a 21-kDa form of LasA, and exhibited high proteolytic and elastolytic activities. Such strain-associated differences in LasA processing and elastase activity can be explained by differences in metal ion-scavenging mechanisms adapted by the strains. Supplementing cultures with calcium stimulated the production of elastase but had no effect on LasA production. The elastase produced exhibited variable sizes, possibly resulting from aberrant processing reactions, and showed little proteolytic activity. Proteolytic activity could be recovered from 33-kDa elastase produced in the presence of calcium by inclusion of zinc in the enzymatic assay. Although iron was previously found to exert a repressive effect on P. aeruginosa elastolytic activity, iron exerted little effect on elastolytic activity when added to cultures containing both zinc and calcium. These studies support the conclusion that elastase production and processing are promoted by both zinc and calcium. LasA production, in comparison, is stimulated by zinc, with both zinc and calcium facilitating its processing. The association of 41-kDa LasA with a low level of elastolytic activity and of 21-kDa LasA with a high level of activity supports the conclusion that lasA encodes a larger, precursor protein which is processed to an active 21-kDa form during secretion.     

Production and properties of an inhibitor of the Pseudomonas autoinducer by Pseudomonas aeruginosa

An inhibitor was found in the culture fluid of Pseudomonas aeruginosa PAO1, which could inhibit the activity of the Pseudomonas autoinducer (PAI). The maximal inhibitory activity occurred in stationary phase culture sup ernatant. The PAI inhibitor did not influence the cell growth and the PAI production by P. aeruginosa PAO1 when the PAI inhibitor was added into culture medium. The induced expression of lacZ in the reporter strain Agrobacterium tumefaciens NT1 was suppressed by this PAI inhibitor, whereas inhibition could be relieved by increasing the auto inducer concentration. The quorum sensing of P. aeruginosa was inhibited presumably by inhibiting the inducing activity of Pseudomonas autoinducer but not by inhibiting the production of Pseudomonas autoinducer. It was demonstrated that the structure of the PAI inhibitor was different from that of acyl-homoserine lactones.     

Regulation of bacterial cell walls: correlation between autolytic activity and cell wall turnover in Staphylococcus aureus.

Cell wall turnover was examined in parent and mutant strains of Staphylococcus aureus. Peptidoglycan and teichoic acid were observed to undergo turnover in the wild-type strain during exponential growth; however, the rate of turnover did not decrease when the growth rate slowed, as the culture entered stationary phase. Isolated native cell walls and crude soluble autolytic enzyme were prepared from cells harvested during exponential and postexponential phases of growth. Native cell walls from both phases of growth autolyzed in buffer at identical rates; similarily, crude soluble enzyme from both preparations degraded radioactive cell walls at the same rate. Therefore, the activity of the autolysin in both exponential and postexponential cells was similar. The autolysis of whole cells of a mutant tar-1 was enhanced by 1.0 M NaCl. When 1.0 M NaCl was present under growing conditions, the rate of cell wall turnover was greatly increased. The presence of chloramphenicol, which inhibits whole-cell autolysis, also inhibited turnover. Analysis of the cell wall material recovered from spent medium revealed products consistent with the known mode of action of the endogenous autolysin. It is concluded that cell wall turnover in S. aureus is independent of the stage of culture growth but is dependent instead on the activity of the autolysin.     

Production and secretion of collagen-binding proteins from Aeromonas veronii

Collagen-binding protein (CNBP) synthesized by Aeromonas veronii is located conserved within the subcellular fraction. The results of this study show that 98% of the total CNBP produced by Aer. veronii is present in the extracellular medium, and that the remaining CNBP is distributed either on the cell surface, within the periplasm or anchored on the outer membrane. CNBP is specifically secreted from Aer. veronii into the culture medium, because all the beta-lactamase activity was located in the cells and could be released by polymixin B extraction of periplasmic proteins. CNBP was produced at growth temperatures from 12 degrees C to 42 degrees C, but not at 4 degrees C. The findings indicate that the level of CNBP in the medium increases during the exponential growth phase and reaches a maximum during the early stationary phase. There was less CNBP production in poor nutrient MMB medium than in the rich LB nutrient medium. CNBP secretion, in contrast to aerolysin secretion, was unaffected by the exeA mutation of Aer. hydrophila. It is concluded that CNBP secretion from Aer. veronii must be achieved by a mechanism different from that reported for aerolysin secretion.     

LytA, major autolysin of Streptococcus pneumoniae, requires access to nascent peptidoglycan

The pneumococcal autolysin LytA is a virulence factor involved in autolysis as well as in fratricidal- and penicillin-induced lysis. In this study, we used biochemical and molecular biological approaches to elucidate which factors control the cytoplasmic translocation and lytic activation of LytA. We show that LytA is mainly localized intracellularly, as only a small fraction was found attached to the extracellular cell wall. By manipulating the extracellular concentration of LytA, we found that the cells were protected from lysis during exponential growth, but not in the stationary phase, and that a defined threshold concentration of extracellular LytA dictates the onset of autolysis. Stalling growth through nutrient depletion, or the specific arrest of cell wall synthesis, sensitized cells for LytA-mediated lysis. Inhibition of cell wall association via the choline binding domain of an exogenously added enzymatically inactive form of LytA revealed a potential substrate for the amidase domain within the cell wall where the formation of nascent peptidoglycan occurs.     

Autolysis of Clostridium acetobutylicum ATCC 824.

The optimum conditions for autolysis of Clostridium acetobutylicum ATCC 824 were determined. Autolysis was optimal at pH 6.3 and 55 degrees C in 0.1 M-sodium acetate/phosphate buffer. The ability of cells to autolyse decreased sharply at the end of the exponential phase of growth. Lysis was stimulated by monovalent cations and compounds that complex divalent cations, and inhibited by divalent cations. The autolysin of C. acetobutylicum, which was mainly cytoplasmic, was purified to homogeneity and characterized as a muramidase. The enzyme was identical to the extracellular muramidase in terms of M(r), isoelectric point and NH2-terminal amino acid sequence. The autolysin was inhibited by lipoteichoic acids and cardiolipin but not by phosphatidylethanolamine and phosphatidylglycerol. A mechanism of regulation and fixation involving lipoteichoic acid, cardiolipin and divalent cations is proposed.     

Purification of a GSH-affinity binding protein from Bacteroides fragilis devoid of glutathione transferase activity

For the location of the aminoglycoside-(3)-N-acetyltransferase isoenzyme II (AAC(3)-II) in the bacterial cell, two strains were studied: Escherichia coli HB101(pJV03), producing the 31-kDa AAC (3)-II enzyme, and E. coli HB101, which served as a control. From each strain five protein fractions were prepared: culture supernatant, and proteins occurring in the periplasm, cytoplasm, inner membrane and outer membrane. All fractions were tested for enzymatic activity of AAC(3)-II. Most of the acetylating activity was found in the cytoplasmic fraction. The distribution of marker enzymes showed a good separation between the periplasmic and the cytoplasmic fraction.     

Protein secretion in Pseudomonas aeruginosa: the xcpA gene encodes an integral inner membrane protein homologous to Klebsiella pneumoniae secretion function protein PulO.

xcp mutations have pleiotropic effects on the secretion of proteins in Pseudomonas aeruginosa PAO. The nucleotide sequence of a 1.2-kb DNA fragment that complements the xcp-1 mutation has been determined. Sequence analysis shows the xcpA gene product to be a 31.8-kDa polypeptide, with a highly hydrophobic character. This is consistent with a localization in the cytoplasmic membrane in P. aeruginosa, determined after specific expression of the xcpA gene under control of the T7 phi 10 promoter. A very strong homology was found between XcpA and PulO, a membrane protein required for pullulanase secretion in Klebsiella pneumoniae. This suggests the existence of a signal sequence-dependent secretion process common to these two unrelated gram-negative bacteria.