The Balance between Protealysin and Its Substrate, the Outer Membrane Protein OmpX, Regulates Serratia proteamaculans Invasion.

Serratia are opportunistic bacteria, causing infections in plants, insects, animals and humans under certain conditions. The development of bacterial infection in the human body involves several stages of host-pathogen interaction, including entry into non-phagocytic cells to evade host immune cells. The facultative pathogen Serratia proteamaculans is capable of penetrating eukaryotic cells. These bacteria synthesize an actin-specific metalloprotease named protealysin. After transformation with a plasmid carrying the protealysin gene, noninvasive E. coli penetrate eukaryotic cells. This suggests that protealysin may play a key role in S. proteamaculans invasion. This review addresses the mechanisms underlying protealysin's involvement in bacterial invasion, highlighting the main findings as follows. Protealysin can be delivered into the eukaryotic cell by the type VI secretion system and/or by bacterial outer membrane vesicles. By cleaving actin in the host cell, protealysin can mediate the reversible actin rearrangements required for bacterial invasion. However, inactivation of the protealysin gene leads to an increase, rather than decrease, in the intensity of S. proteamaculans invasion. This indicates the presence of virulence factors among bacterial protealysin substrates. Indeed, protealysin cleaves the virulence factors, including the bacterial surface protein OmpX. OmpX increases the expression of the EGFR and ß1 integrin, which are involved in S. proteamaculans invasion. It has been shown that an increase in the invasion of genetically modified S. proteamaculans may be the result of the accumulation of full-length OmpX on the bacterial surface, which is not cleaved by protealysin. Thus, the intensity of the S. proteamaculans invasion is determined by the balance between the active protealysin and its substrate OmpX.

Cold-adapted strains as plant growth-promoting bacteria on soybean seeds and biocontrol agents against Macrophomina phaseolina.

AIM: The aim was to characterize cold-adapted bacteria by testing their PGP features and antagonistic activity against Macrophomina phaseolina, both in vitro and coating soybean seeds (Glycine max [L.] Merr.). METHODS AND RESULTS: Burkholderia gladioli MB39, Serratia proteamaculans 136 and Serratia proteamaculans 137 were evaluated. In vitro tests showed that S. proteamaculans 136 and 137 produce siderophore and indole-acetic acid (IAA), solubilize phosphate and fix nitrogen. Additionally, B. gladioli MB39 and S. proteamaculans 137 showed hydrolase activity and potent antifungal effects. The biocontrol efficacy over soybean seeds was evaluated using in vitro and greenhouse methods by immersing seeds into each bacterial suspension. As a result, S. proteamaculans 136 has improved the performance in all the seed germination evaluated parameters. In addition, S. proteamaculans 137 and B. gladioli MB39 strongly inhibited M. phaseolina, reducing the infection index values to 10% and 0%, respectively. CONCLUSION: Serratia proteamaculans 136, 137 and Burkholderia gladioli MB39 showed plant growth promotion features and inhibition of Macrophomina phaseolina infection by producing different antifungal compounds. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results reinforce the application of cold-adapted Serratia proteamaculans and Burkholderia gladioli bacterial strains as candidates for developing microbial formulation to promote plant growth and guarantee antifungal protection in soybean crops.

Protealysin Targets the Bacterial Housekeeping Proteins FtsZ and RecA.

Serratia proteamaculans synthesizes the intracellular metalloprotease protealysin. This work was aimed at searching for bacterial substrates of protealysin among the proteins responsible for replication and cell division. We have shown that protealysin unlimitedly cleaves the SOS response protein RecA. Even 20% of the cleaved RecA in solution appears to be incorporated into the polymer of uncleaved monomers, preventing further polymerization and inhibiting RecA ATPase activity. Transformation of Escherichia coli with a plasmid carrying the protealysin gene reduces the bacterial UV survival up to 10 times. In addition, the protealysin substrate is the FtsZ division protein, found in both E. coli and Acholeplasma laidlawii, which is only 51% identical to E. coli FtsZ. Protealysin cleaves FtsZ at the linker between the globular filament-forming domain and the C-terminal peptide that binds proteins on the bacterial membrane. Thus, cleavage of the C-terminal segment by protealysin can lead to the disruption of FtsZ's attachment to the membrane, and thereby inhibit bacterial division. Since the protealysin operon encodes not only the protease, but also its inhibitor, which is typical for the system of interbacterial competition, we assume that in the case of penetration of protealysin into neighboring bacteria that do not synthesize a protealysin inhibitor, cleavage of FtsZ and RecA by protealysin may give S. proteamaculans an advantage in interbacterial competition.

Bacterial Outer Membrane Protein OmpX Regulates ß1 Integrin and Epidermal Growth Factor Receptor (EGFR) Involved in Invasion of M-HeLa Cells by Serratia proteamaculans.

Opportunistic pathogen Serratia proteamaculans are able to penetrate the eukaryotic cells. The penetration rate can be regulated by bacterial surface protein OmpX. OmpX family proteins are able to bind to host cell surface to the epidermal growth factor receptor (EGFR) and the extracellular matrix protein fibronectin, whose receptors are in return the α5 ß1 integrins. Here we elucidated the involvement of these host cell proteins in S. proteamaculans invasion. We have shown that, despite the absence of fibronectin contribution to S. proteamaculans invasion, ß1 integrin was directly involved in invasion of M-HeLa cells. Herewith ß1 integrin was not the only receptor that determines sensitivity of host cells to bacterial invasion. Signal transfer from EGFR was also involved in the penetration of these bacteria into M-HeLa cells. However, M-HeLa cells have not been characterized by large number of these receptors. It turned out that S. proteamaculans attachment to the host cell surface resulted in an increment of EGFR and ß1 integrin genes expression. Such gene expression increment also caused Escherichia coli attachment, transformed with a plasmid encoding OmpX from S. proteamaculans. Thus, an OmpX binding to the host cell surface caused an increase in the EGFR and ß1 integrin expression involved in S. proteamaculans invasion.

Serratia proteamaculans Strain AGR96X Encodes an Antifeeding Prophage (Tailocin) with Activity against Grass Grub (Costelytra giveni) and Manuka Beetle (Pyronota Species) Larvae.

A highly virulent Serratia proteamaculans strain, AGR96X, exhibiting specific pathogenicity against larvae of the New Zealand grass grub (Costelytra giveni; Coleoptera: Scarabaeidae) and the New Zealand manuka beetle (Pyronota festiva and P. setosa; Coleoptera: Scarabaeidae), was isolated from a diseased grass grub larva. A 12-day median lethal dose of 4.89 × 103 ± 0.92 × 103 cells per grass grub larva was defined for AGR96X, and death occurred within 5 to 12 days following the ingestion of a high bacterial dose. During the infection period, the bacterium rapidly multiplied within the insect host and invaded the hemocoel, leading to a mean bacterial load of 8.2 × 109 cells per larva at 6 days postingestion. Genome sequencing of strain AGR96X revealed the presence of a variant of the Serratia entomophila antifeeding prophage (Afp), a tailocin designated AfpX. Unlike Afp, AfpX contains two Afp16 tail-length termination protein orthologs and two putative toxin components. A 37-kb DNA fragment encoding the AfpX-associated region was cloned, transformed into Escherichia coli, and fed to C. giveni and Pyronota larvae, causing mortality. In addition, the deletion of the afpX15 putative chaperone component abolished the virulence of AGR96X. Unlike S. entomophila Afp, the AfpX tailocin could be induced by mitomycin C. Transmission electron microscopy analysis revealed the presence of Afp-like particles of various lengths, and when the purified AfpX tailocin was fed to grass grub or manuka beetle larvae, they underwent phenotypic changes similar to those of larvae fed AGR96X.IMPORTANCESerratia proteamaculans strain AGR96X shows dual activity against larvae of endemic New Zealand pasture pests, the grass grub (Costelytra giveni) and the manuka beetle (Pyronota spp.). Unlike Serratia entomophila, the causal agent of amber disease, which takes 3 to 4 months to kill grass grub larvae, AGR96X causes mortality within 5 to 12 days of ingestion and invades the insect hemocoel. AGR96X produces a unique variant of the S. entomophila antifeeding prophage (Afp), a cell-free phage-like entity that is proposed to deliver protein toxins to the grass grub target site, causing a cessation of feeding activity. Unlike other Afp variants, AGR96X Afp, named AfpX, contains two tail-length termination proteins, resulting in greater variability in the AfpX length. AfpX shows dual activity against both grass grub and manuka beetle larvae. AGR96X is a viable alternative to S. entomophila for pest control in New Zealand pasture systems.

Cloning, sequencing, expression, and characterization of thermostability of oligopeptidase B from Serratia proteamaculans, a novel psychrophilic protease.

Protease from Serratia proteamaculans (PSP) is the first known psychrophilic oligopeptidase B. The gene of S. proteamaculans 94 oligopeptidase B was cloned, sequenced and expressed in Escherichia coli. The unfolding of PSP molecule following heat treatment at 37°C by measuring fluorescence spectra was examined in parallel with the residual activity determination. The effect of PSP thermostabilization by glycerol at 37-50 °Ð¡ was revealed. Calcium ions and buffer solution of low molarity cause the opposite effect - the acceleration of PSP inactivation at 37°C. The thermal stability of PSP molecule in the presence of 0-100mM CaCl2 was also investigated by means of high-sensitivity differential scanning calorimetry. The artificial reconstruction of the natural complex PSP-chaperonin from S. рroteamaculans was carried out: the stable complex (1:1) of chaperonin E. сoli GroEL with active recombinant enzyme PSP was obtained. It was shown that complex formation with chaperonin promotes PSP thermostability at 37°C.

The role of the chi1 gene from the endophytic bacteria Serratia proteamaculans 336x in the biological control of wheat take-all.

Take-all, a disease caused by the fungus Gaeumannomyces graminis var. tritici, is the most important root disease of wheat and causes severe yield losses worldwide. Using microorganisms as biological agents to control the disease is important because no resistant cultivars or effective chemical fungicides are available. In this study, we tested the biological control capability of a chitinase produced by the endophytic bacterium Serratia proteamaculans 336x against wheat take-all. The chitinase gene chi1 of S. proteamaculans 336x was cloned and heterologously expressed in Escherichia coli. The recombinant protein exhibited chitinase activity and in vitro antifungal activity against G. graminis var. tritici. With in-frame deletion of the chi1 gene by homologous recombination, the chi1-deleted mutant was devoid of chitinase activity and the biocontrol efficacy was reduced by 42.5%. The complementation of the Δchi1 mutant strain by the chi1 gene resulted in the partial restoration of the chitinase activity and biocontrol efficacy. These results support a role for the Chi1 protein in the biocontrol process of S. proteamaculans 336x against wheat take-all.

From biomolecules to biogeochemistry: Exploring the interaction of an indigenous bacterium with gold.

Specialised microbial communities colonise the surface of gold particles in soils/sediments, and catalyse gold dissolution and re-precipitation, thereby contributing to the environmental mobility and toxicity of this 'inert' precious metal. We assessed the proteomic and physiological response of Serratia proteamaculans, the first metabolically active bacterium enriched and isolated directly from natural gold particles, when exposed to toxic levels of soluble Au3+ (10 µM). The results were compared to a metal-free blank, and to cultures exposed to similarly toxic levels of soluble Cu2+ (0.1 mM); Cu was chosen for comparison because it is closely associated with Au in nature due to similar geochemical properties. A total of 273 proteins were detected from the cells that experienced the oxidative effects of soluble Au, of which 139 (51%) were upregulated with either sole expression (31%) or had synthesis levels greater than the Au-free control (20%). The majority (54%) of upregulated proteins were functionally different from up-regulated proteins in the bacteria-copper treatment. These proteins were related to broad functions involving metabolism and biogenesis, followed by cellular process and signalling, indicating significant specificity for Au. This proteomic study revealed that the bacterium upregulates the synthesis of various proteins related to oxidative stress response (e.g., Monothiol-Glutaredoxin, Thiol Peroxidase, etc.) and cellular damage repair, which leads to the formation of metallic gold nanoparticles less toxic than ionic gold. Therefore, indigenous bacteria may mediate the toxicity of Au through two different yet simultaneous processes: i) repairing cellular components by replenishing damaged proteins and ii) neutralising reactive oxygen species (ROS) by up-regulating the synthesis of antioxidants. By connecting the fields of molecular bacteriology and environmental biogeochemistry, this study is the first step towards the development of biotechnologies based on indigenous bacteria applied to gold bio-recovery and bioremediation of contaminated environments.

Fatal Bronchopneumonia and Tracheitis in a Green Turtle (Chelonia mydas) Caused by Serratia proteamaculans.

A free-ranging subadult, male green turtle (Chelonia mydas) presented with radiographic evidence of pneumonia and died acutely. On necropsy, the trachea and bronchi were plugged by diphtheritic membranes, comprised of fibrin, necrotic debris, and colonies of bacilli, identified as Serratia proteamaculans. S. proteamaculans, typically considered an opportunistic plant pathogen, has rarely been described as causing disease in animals. This is the first report of S. proteamaculans causing severe necrotizing tracheitis and bronchopneumonia in a reptile.

Identification and Characterization of a New Serratia proteamaculans Strain That Naturally Produces Significant Amount of Extracellular Laccase.

Natural biodegradation processes hold promises for the conversion of agro-industrial lignocellulosic biomaterials into biofuels and fine chemicals through lignin-degrading enzymes. The high cost and low stability of these enzymes remain a significant challenge to economic lignocellulosic biomass conversion. Wood-degrading microorganisms are a great source for novel enzyme discoveries. In this study, the decomposed wood samples were screened, and a promising γ-proteobacterial strain that naturally secreted a significant amount of laccase enzyme was isolated and identified as Serratia proteamaculans AORB19 based on its phenotypic and genotypic characteristics. The laccase activities in culture medium of strain AORB19 were confirmed both qualitatively and quantitatively. Significant cultural parameters for laccase production under submerged conditions were identified following a one-factor-at-a-time (OFAT) methodology: temperature 30°C, pH 9, yeast extract (2 g/l), Li+, Cu2+, Ca2+, and Mn2+ (0.5 mM), and acetone (5%). Under the selected conditions, a 6-fold increase (73.3 U/L) in laccase production was achieved when compared with the initial culturing conditions (12.18 U/L). Furthermore, laccase production was enhanced under alkaline and mesophilic growth conditions in the presence of metal ions and organic solvents. The results of the study suggest the promising potential of the identified strain and its enzymes in the valorization of lignocellulosic wastes. Further optimization of culturing conditions to enhance the AORB19 strain laccase secretion, identification and characterization of the purified enzyme, and heterologous expression of the specific enzyme may lead to practical industrial and environmental applications.

Identification of Diverse Toxin Complex Clusters and an eCIS Variant in Serratia proteamaculans Pathovars of the New Zealand Grass Grub (Costelytra Giveni) and Manuka Beetle (Pyronota Spp.) Larvae.

The grass grub endemic to New Zealand, Costelytra giveni (Coleoptera: Scarabaeidae), and the manuka beetle, Pyronota festiva and P. setosa (Coleoptera: Scarabaeidae), are prevalent pest species. Through assessment of bacterial strains isolated from diseased cadavers of these insect species, 19 insect-active Serratia proteamaculans variants and a single Serratia entomophila strain were isolated. When independently bioassayed, these isolates differed in host range, the rate of disease progression, and 12-day mortality rates, which ranged from 60 to 100% of the challenged larvae. A Pyronota spp.-derived S. proteamaculans isolate caused a transient disease phenotype in challenged C. giveni larvae, whereby larvae appeared diseased before recovering to a healthy state. Genome sequence analysis revealed that all but two of the sequenced isolates contained a variant of the S. entomophila amber-disease-associated plasmid, pADAP. Each isolate also encoded one of seven distinct members of the toxin complex (Tc) family of insect-active toxins, five of which are newly described, or a member of the extracellular contractile injection (eCIS) machine family, with a new AfpX variant designated SpF. Targeted mutagenesis of each of the predicted Tc- or eCIS-encoding regions abolished or attenuated pathogenicity. Host-range testing showed that several of the S. proteamaculans Tc-encoding isolates affected both Pyronota and C. giveni species, with other isolates specific for either Pyronota spp. or C. giveni. The isolation of several distinct host-specific pathotypes of Serratia spp. may reflect pathogen-host speciation. IMPORTANCE New pathotypes of the insect pathogen Serratia, each with differing virulence attributes and host specificity toward larvae of the New Zealand manuka beetle and grass grub, have been identified. All of the Serratia proteamaculans isolates contained one of seven different insect-active toxin clusters or one of three eCIS variants. The diversity of these Serratia-encoded virulence clusters, resulting in differences in larval disease progression and host specificity in endemic scarab larvae, suggests speciation of these pathogens with their insect hosts. The differing virulence properties of these Serratia species may affect their potential infectivity and distribution among the insect populations. Based on their differing geographic isolation and pathotypes, several of these Serratia isolates, including the manuka beetle-active isolates, are likely to be more effective biopesticides in specific environments or could be used in combination for greater effect.

The Role of SprIR Quorum Sensing System in the Regulation of Serratia proteamaculans 94 Invasion.

The bacteria Serratia proteamaculans 94 have a LuxI/LuxR type QS system consisting of AHL synthase SprI and the regulatory receptor SprR. We have previously shown that inactivation of the AHL synthase sprI gene resulted in an increase in the invasive activity of S. proteamaculans correlated with an increased bacterial adhesion. In the present work, the effects of inactivation of the S. proteamaculans receptor SprR are studied. Our results show that inactivation of the receptor sprR gene leads to an increase in bacterial invasion without any increase in their adhesion. On the other hand, inactivation of the sprR gene increases the activity of the extracellular protease serralysin. Inactivation of the QS system does not affect the activity of the pore-forming toxin ShlA and prevents the ShlA activation under conditions of a limited concentration of iron ions typical of the human body. While the wild type strain shows increased invasion in the iron-depleted medium, deletion of its QS system leads to a decrease in host cell invasion, which is nevertheless similar to the level of the wild type S. proteamaculans grown in the iron-rich medium. Thus, inactivation of either of the two component of the S. proteamaculans LuxI/LuxR-type QS system leads to an increase in the invasive activity of these bacteria through different mechanisms and prevents invasion under the iron-limited conditions.

NMR assignments and secondary structure distribution of emfourin, a novel proteinaceous protease inhibitor.

Emfourin (M4in) from Serratia proteamaculans is a new proteinaceous inhibitor of protealysin-like proteases (PLPs), a subgroup of the well-known and widely represented metallopeptidase M4 family. Although the biological role of PLPs is debatable, data published indicate their involvement in pathogenesis, including bacterial invasion into eukaryotic cells, suppression of immune defense of some animals, and destruction of plant cell walls. Gene colocalization into a bicistronic operon observed for some PLPs and their inhibitors (as in the case of M4in) implies a mutually consistent functioning of both entities. The originality of the amino acid sequence of M4in suggests it belongs to a previously unknown protein family and this encourages structural studies. In this work, we report a near-complete assignment of 1H, 13C, and 15N resonances of recombinant M4in and its structural-dynamic properties derived from the chemical shifts. According the NMR data analysis, the M4in molecule comprises 3-5 helical elements and 4-6 ß-strands, at least two of which are apparently antiparallel, ascribing this obviously globular protein to the α + ß structural class. Besides, two disordered regions also exist in the central loops between the regular secondary structural elements. The obtained data provide the basis for determining the high-resolution structure as well as functioning mechanism of M4in that can be used for development of new antibacterial therapeutic strategies.

Invasion of Serratia proteamaculans is regulated by the sprI gene encoding AHL synthase.

Quorum Sensing (QS) system regulates gene expression in response to a change in the density of the bacterial population. Facultative pathogen Serratia proteamaculans 94 has a LuxI/LuxR type QS system consisting of regulatory protein SprR and AHL synthase SprI. Invasive activity of these bacteria appears at the stationary growth phase corresponding to a maximal density of the bacterial population in vitro. To evaluate the contribution of QS system of S. proteamaculans 94 to the regulation of invasive activity, in this work, S. proteamaculans SprI(-) mutant carrying the inactivated AHL synthase gene was used. Inactivation of the AHL synthase sprI gene resulted in a more than fourfold increase in the invasive activity of S. proteamaculans preceded by the increased adhesion of bacteria to the cell surface. This effect correlated with the increased expression of the outer membrane protein ompX gene and the decrease in the activity of intrabacterial protease protealysin, whose substrate is OmpX. The inverse correlation between activity of protealysin and bacterial invasion was also observed in the model experiments under the iron-limiting culture conditions. These results show that QS system regulates the S. proteamaculans invasion. This regulation can involve changes both in the protealysin activity and in the level of the ompX gene transcription.

Structural inspection and protein motions modelling of a fungal glycoside hydrolase family 18 chitinase by crystallography depicts a dynamic enzymatic mechanism.

Chitinases degrade chitin into low molecular weight chitooligomers, which have a broad range of industrial, agricultural, and medical functions. Understanding the relationship between the diverse characteristics of chitinases and their functions is necessary for the improvement of functional enzymes that meet specific requirements. We report here a full crystallographic analysis of three complexes obtained from the chitinase Chit42 from Trichoderma harzianum, which represent different states along the enzymatic mechanism. The inactive double mutant D169A/E171A was submitted to soaking/crystallization experiments with hexa-N-acetyl-glucosamine (NAG6) or tetra-N-acetyl-glucosamine (NAG4), trapping the enzyme-substrate complex (Chit42-NAG6), the enzyme-products complex (Chit42-NAG4-NAG2) and a someway intermediate state. Structural comparison among the different complexes depicts the determinants defining the different subsites and revealed a previously unobserved dynamic on-off ligand binding process associated with a motion of its insertion domain, which might be accompanying the role or aromatics in processivity. An ensemble refinement performed to extract dynamic details from the diffraction data elucidates the implication of some highly flexible residues in the productive sliding of the substrate and the product release event. These positions were submitted to mutagenesis and the activity of the variants was investigated in the hydrolysis of NAG6, colloidal chitin and two chitosans with different polymerization and acetylation degree. All the changes affected the Chit42 hydrolytic activity therefore confirming the involvement of these positions in catalysis. Furthermore, we found the variants R295S and E316S improving the apparent catalytic efficiency of chitin and NAG6 and, together with E316A, enhancing the specific activity on chitosan. Therefore, our results provide novel insight into the molecular mechanisms underlying the hydrolysis of chitinous material by fungal chitinases, and suggest new targets to address engineering of these biotechnologically important enzymes.

Cleavage of the outer membrane protein OmpX by protealysin regulates Serratia proteamaculans invasion.

Protealysin is a thermolysin-like protease of Serratia proteamaculans capable of specifically cleaving actin, which correlates with the invasive activity of these bacteria. Here, we show that inactivation of the protealysin gene does not inhibit invasion but, in contrast, leads to a twofold increase in the S. proteamaculans invasive activity. By mass spectrometry, we identified the outer membrane protein OmpX as a substrate of protealysin. Recombinant E. coli carrying the OmpX gene truncated by 40 N-terminal residues or both the OmpX and protealysin genes, in contrast to the full-length OmpX, do not increase adhesion of these bacteria, indicating that the 40 N-terminal residues of OmpX are indispensable for S. proteamaculans invasion. Our results show that both protealysin and its substrates can stimulate Serratia invasion.

Molecular dynamics complemented by site-directed mutagenesis reveals significant difference between the interdomain salt bridge networks stabilizing oligopeptidases B from bacteria and protozoa in their active conformations.

Oligopeptidases B (OpdBs) are trypsin-like peptidases from protozoa and bacteria that belong to the prolyl oligopeptidase (POP) family. All POPs consist of C-terminal catalytic domain and N-terminal ß-propeller domain and exist in two major conformations: closed (active), where the domains and residues of the catalytic triad are positioned close to each other, and open (non-active), where two domains and residues of the catalytic triad are separated. The interdomain interface, particularly, one of its salt bridges (SB1), plays a role in the transition between these two conformations. However, due to double amino acid substitution (E/R and R/Q), this functionally important SB1 is absent in γ-proteobacterial OpdBs including peptidase from Serratia proteamaculans (PSP). In this study, molecular dynamics was used to analyze inter- and intradomain interactions stabilizing PSP in the closed conformation, in which catalytic H652 is located close to other residues of the catalytic triad. The 3D models of either wild-type PSP or of mutant PSPs carrying activating mutations E125A and D649A in complexes with peptide-substrates were subjected to the analysis. The mechanism that regulates transition of H652 from active to non-active conformation upon domain separation in PSP and other γ-proteobacterial OpdB was proposed. The complex network of polar interactions within H652-loop/C-terminal α-helix and between these areas and ß-propeller domain, established in silico, was in a good agreement with both previously published results on the effects of single-residue mutations and new data on the effects of the activating mutations on each other and on the low active mutant PSP-K655A.Communicated by Ramaswamy H. Sarma.

Protealysin is not Secreted Constitutively.

BACKGROUND: Protealysin, a zinc metalloprotease of Serratia proteamaculans, is the prototype of a new group within the peptidase family M4. Protealysin-like proteases (PLPs) are widely spread in bacteria but are also found in fungi and archaea. The biological functions of PLPs have not been well studied, but published data showed the involvement of enzymes of this group in the interaction of bacteria with higher organisms, and most likely in the pathogenesis. Such functionality requires the release of the proteases from bacterial cells; however, the data on the cellular localization of PLPs are contradictory and no direct data of this kind have been published. OBJECTIVE: Here, the protealysin cellular localization was studied for the first time using immunochemical methods. METHODS AND RESULTS: We have produced polyclonal rabbit antibodies against the protealysin precursor. The enzyme was evaluated in cells and medium of periodic culture of S. proteamaculans 94 using Western blotting as well as the enzyme localization was analysed by immunoelectron microscopy. It was shown that more than 99% of the enzyme is in a cell-associated form. Protealysin is accumulated in cells as an inactive precursor. It matures only after the release from cells (after their lysis). Immunoelectron microscopy analysis of bacterial cells has revealed no specific localization of protealysin; it was evenly distributed in the cytoplasm. CONCLUSION: The data obtained suggest that S. proteamaculans protealysin and supposedly other protealysin-like proteases are not secreted constitutively and their release from bacteria is likely induced by a certain stimulus such as a contact with a eukaryotic cell. This finding is critical for further studies of the involvement of these enzymes in pathogenesis.

Reversible Cyclic Thermal Inactivation of Oligopeptidase B from Serratia proteamaculans.

A unique property was found for oligopeptidase B from Serratia proteamaculans (PSP) as well as its mutants: they can undergo reversible thermal inactivation at 37°C, with activity being restored or even increased with respect to the initial one upon subsequent cooling. The process can be repeated several times, with the same results achieved (up to 5 cycles). This effect can be explained by a shift in the equilibrium between the inactive open form of the enzyme and the active closed one upon variation of the incubation temperature.

Activity modulation of the oligopeptidase B from Serratia proteamaculans by site-directed mutagenesis of amino acid residues surrounding catalytic triad histidine.

Oligopeptidase B (OpdB; EC 3.4.21.83) is a trypsin-like peptidase belonging to the family of serine prolyl oligopeptidases; two-domain structure of the enzyme includes C-terminal peptidase catalytic domain and N-terminal seven-bladed ß-propeller domain. Importance of the interface between these domains and particularly of the 5 salt bridges for enzyme activity was established for protozoan OpdBs. However, these salt bridges are not conserved in γ -proteobacterial OpdBs including the peptidase from Serratia proteamaculans (PSP). In this work, using comparative modelling and protozoan OpdBs' crystal structures we created 3D models of PSP in open and closed forms to elucidate the mechanism underlying inactivation of the truncated form of PSP1-655 obtained earlier. Analysis of the models shows that in the closed form of PSP charged amino acid residues of histidine loop, surrounding the catalytic triad His652, participate in formation of the inter-domain contact interface between catalytic and ß-propeller domains, while in the open form of PSP disconnection of the catalytic triad and distortion of these contacts can be observed. Complete destruction of this interface by site-directed mutagenesis causes inactivation of PSP while elimination of the individual contacts leads to differential effects on the enzyme activity and substrate specificity. Thus, we identified structural factors regulating activity of PSP and supposedly of other γ-proteobacterial OpdBs and discovered the possibility of directed modulation of their enzymatic features.