Serratia Proteamaculans Research Articles

Psychrophile spoilers dominate the bacterial microbiome in musculature samples of slaughter pigs

The aim of this study was to disentangle the microbial diversity on porcine musculature. The hypervariable V1–V2 region of the 16S rRNA gene was amplified from DNA samples of clinically healthy slaughter pigs (n=8). Pyrosequencing yielded 37,000 quality-controlled reads and a diverse microbiome with 54–159 OTUs per sample was detected. Interestingly, 6 out of 8 samples were strongly dominated by 1–2 highly abundant OTUs (best hits of highly abundant OTUs: Serratia proteamaculans, Pseudomonas syringae, Aeromonas allosaccharophila, Brochothrix thermosphacta, Acidiphilium cryptum and Escherichia coli). In 1g musculature scraping, 3.20E+06 16S rRNA gene copies and 4.45E+01 Enterobacteriaceae rRNA gene copies were detected with qPCR. We conclude that i.) next-generation sequencing technologies help encompass the full content of complex, bacterial contamination, ii.) psychrophile spoilers dominated the microbiota and iii.) E. coli is an effective marker species for pork contamination, as it was one of very few abundant species being present in all samples.

Detection and Molecular Identification of Persistent Water Vessel Colonizing Bacteria in a Table Water Factory in Nigeria

Aims: To establish organisms persistent in a table water production facility and to determine the points they entered in order to eliminate any risk of having pathogens in a water production system. Study Design: Analytic observational studies Place and Duration of Study: Bottling facility in Nigeria and University of Nottingham, United Kingdom. Study was between August 2011 and Sept 2013. Methodology: Sample control points were subjected to counts of bacteria, yeasts, and mold using membrane filtration to trace the source of increased bacteria counts in a table water production factory. Organisms were identified by 16S rRNA sequencing and biofilm formation was assessed Original Research Article Nwaiwu and Nwachukwu; BMRJ, 13(5): 1-12, 2016; Article no.BMRJ.24378 2 with micro titer dish biofilm formation analysis. Results: Total bacteria and Pseudomonas Spp. count were highest in the carbon filter and 5% (v/v) chlorine used for disinfection was found to be effective against planktonic cells of 18 hour cultures of Pseudomonas Spp. isolated from the carbon filter tank. No yeasts and mold were detected and after a sand blasting exercise to clean affected tanks, total bacteria counts on tryptone glucose extract medium decreased to less than factory allowable limits of 25 cfu per 100 ml of water with no further growth of Pseudomonas Spp. on centrimide medium. Other bacteria that emerged after the sand blasting exercise were regarded as persistent but had less biofilm forming ability (p=.02) when compared with Pseudomonas aeruginosa PAO1. The persistent bacteria identified after sequencing were Aeromonas hydrophila and Serratia proteamaculans. Conclusion: Apart from Pseudomonas Spp., other bacteria can persist in the water tanks of a water bottling facility and routine checks may fail to detect an underlying problem until it becomes obvious. Prompt corrective action ensures that public safety is not compromised. This was the first time Aeromonas hydrophila and Serratia proteamaculans were identified from the water tanks in the bottling facility.

The Metabolically Active Bacterial Microbiome of Tonsils and Mandibular Lymph Nodes of Slaughter Pigs.

The exploration of microbiomes in lymphatic organs is relevant for basic and applied research into explaining microbial translocation processes and understanding cross-contamination during slaughter. This study aimed to investigate whether metabolically active bacteria (MAB) could be detected within tonsils and mandibular lymph nodes (MLNs) of pigs. The hypervariable V1-V2 region of the bacterial 16S rRNA genes was amplified from cDNA from tonsils and MLNs of eight clinically healthy slaughter pigs. Pyrosequencing yielded 82,857 quality-controlled sequences, clustering into 576 operational taxonomic units (OTUs), which were assigned to 230 genera and 16 phyla. The actual number of detected OTUs per sample varied highly (23–171 OTUs). Prevotella zoogleoformans and Serratia proteamaculans (best type strain hits) were most abundant (10.6 and 41.8%, respectively) in tonsils and MLNs, respectively. To explore bacterial correlation patterns between samples of each tissue, pairwise Spearman correlations (rs) were calculated. In total, 194 strong positive and negative correlations |rs| ≥ 0.6 were found. We conclude that (i) lymphatic organs harbor a high diversity of MAB, (ii) the occurrence of viable bacteria in lymph nodes is not restricted to pathological processes and (iii) lymphatic tissues may serve as a contamination source in pig slaughterhouses. This study confirms the necessity of the EFSA regulation with regard to a meat inspection based on visual examinations to foster a minimization of microbial contamination.

Serratia aquatilis sp. nov., isolated from drinking water systems.

A cream-white-pigmented, oxidase-negative bacterium (strain 2015-2462-01T), isolated from a drinking water system, was investigated in detail to determine its taxonomic position. Cells of the isolate were rod-shaped and stained Gram-negative. A comparison of the 16S rRNA gene sequence of strain 2015-2462-01T with sequences of the type strains of closely related species of the genus Serratia revealed highest similarity to Serratia fonticola (98.4 %), Serratia proteamaculans (97.8 %), Serratia liquefaciens and Serratia grimesii (both 97.7 %). 16S rRNA gene sequence similarities to all other Serratia species were below 97.4 %. Multilocus sequence analysis (MLSA) on the basis of concatenated partial gyrB, rpoB, infB and atpD gene sequences showed a clear distinction of strain 2015-2462-01T from the type strains of the closest related Serratia species. The fatty acid profile of the strain consisted of C16 : 1 ω7c, C16 : 0; C14 : 0 and C14 : 0 3-OH/iso-C16 : 1 I as major components. DNA-DNA hybridizations between 2015-2462-01T and S. fonticola ATCC 29844T resulted in a relatedness value of 27 % (reciprocal 20 %). This DNA-DNA hybridization result in combination with the MLSA results and the differential biochemical properties indicated that strain 2015-2462-01T represents a novel species of the genus Serratia, for which the name Serratia aquatilis sp. nov. is proposed. The type strain is 2015-2462-01T ( = LMG 29119T = CCM 8626T).

Inverse relationship between chitobiase and transglycosylation activities of chitinase-D from Serratia proteamaculans revealed by mutational and biophysical analyses.

Serratia proteamaculans chitinase-D (SpChiD) has a unique combination of hydrolytic and transglycosylation (TG) activities. The TG activity of SpChiD can be used for large-scale production of chito-oligosaccharides (CHOS). The multiple activities (hydrolytic and/or chitobiase activities and TG) of SpChiD appear to be strongly influenced by the substrate-binding cleft. Here, we report the unique property of SpChiD substrate-binding cleft, wherein, the residues Tyr28, Val35 and Thr36 control chitobiase activity and the residues Trp160 and Trp290 are crucial for TG activity. Mutants with reduced (V35G and T36G/F) or no (SpChiDΔ30–42 and Y28A) chitobiase activity produced higher amounts of the quantifiable even-chain TG product with degree of polymerization (DP)-6, indicating that the chitobiase and TG activities are inversely related. In addition to its unprecedented catalytic properties, unlike other chitinases, the single modular SpChiD showed dual unfolding transitions. Ligand-induced thermal stability studies with the catalytically inactive mutant of SpChiD (E153A) showed that the transition temperature increased upon binding of CHOS with DP2–6. Isothermal titration calorimetry experiments revealed the exceptionally high binding affinities for E153A to CHOS with DP2–6. These observations strongly support that the architecture of SpChiD substrate-binding cleft adopted to control chitobiase and TG activities, in addition to usual chitinase-mediated hydrolysis.

Truncated Variants of Serratia proteamaculans Oligopeptidase B Having Different Activities.

Treatment of native psychrophilic oligopeptidase B from Serratia proteamaculans (PSP, 78 kDa) with chymotrypsin (soluble or immobilized on modified porous glass MPG-PA) in the presence of 50% glycerol leads to production of a truncated enzyme form (PSP-Chtr, ~66 kDa), which retains activity toward the low molecular weight substrate of PSP, BAPNA, but in contrast to PSP, is active toward the protein substrate azocasein. It has been shown by MALDI-TOF mass-spectrometry that PSP-Chtr lacks the N-terminal region of the molecule that envelops the catalytic domain of PSP and supposedly prevents hydrolysis of high molecular weight substrates. It has also been established that the lacking fragment corresponds to the N-terminal highest rank element of the informational structure of PSP. This finding confirms the usefulness of the method of informational structure analysis for protein engineering of enzymes. A similar treatment of PSP with immobilized trypsin also led to production of a stable truncated enzyme form (PSP-Tr, ~75kDa) which lacked 22 C-terminal amino acid residues and completely lost enzymatic activity, presumably because of changes in the nearest environment of His652 of the catalytic triad.

Production of bioactive chitosan oligosaccharides using the hypertransglycosylating chitinase-D from Serratia proteamaculans

The biological activities of chitosan and its oligosaccharides are greatly influenced by properties such as the degree of polymerization (DP), degree of acetylation (DA) and pattern of acetylation (PA). Here, structurally diverse chitosan oligosaccharides from chitosan polymers (DA=35% or 61%) were generated using Serratia proteamaculans wild-type chitinase D (SpChiD) and the W114A mutant which lacks transglycosylase activity. The crude oligosaccharide mixtures and purified fractions with specific DP and DA ranges were tested for their ability to induce an oxidative burst in rice cell suspension cultures. The crude mixtures were more active when produced by the W114A mutant whereas the purified fractions were more active when produced by wild-type SpChiD. Neither hydrolysis nor transglycosylation by SpChiD was inhibited in the presence of fully-deacetylated oligosaccharides, suggesting that SpChiD could be exploited to generate oligosaccharides with defined DA and PA values.

Transcriptomic changes in the plant pathogenic fungus Rhizoctonia solani AG-3 in response to the antagonistic bacteria Serratia proteamaculans and Serratia plymuthica.

BackgroundImproved understanding of bacterial-fungal interactions in the rhizosphere should assist in the successful application of bacteria as biological control agents against fungal pathogens of plants, providing alternatives to chemicals in sustainable agriculture. Rhizoctonia solani is an important soil-associated fungal pathogen and its chemical treatment is not feasible or economic. The genomes of the plant-associated bacteria Serratia proteamaculans S4 and Serratia plymuthica AS13 have been sequenced, revealing genetic traits that may explain their diverse plant growth promoting activities and antagonistic interactions with R. solani. To understand the functional response of this pathogen to different bacteria and to elucidate whether the molecular mechanisms that the fungus exploits involve general stress or more specific responses, we performed a global transcriptome profiling of R. solani Rhs1AP anastomosis group 3 (AG-3) during interaction with the S4 and AS13 species of Serratia using RNA-seq.ResultsApproximately 104,504 million clean 75-100 bp paired-end reads were obtained from three libraries, each in triplicate (AG3-Control, AG3-S4 and AG3-AS13). Transcriptome analysis revealed that approximately 10 % of the fungal transcriptome was differentially expressed during challenge with Serratia. The numbers of S4- and AS13-specific differentially expressed genes (DEG) were 866 and 292 respectively, while there were 1035 common DEGs in the two treatment groups. Four hundred and sixty and 242 genes respectively had values of log2 fold-change > 3 and for further analyses this cut-off value was used. Functional classification of DEGs based on Gene Ontology enrichment analysis and on KEGG pathway annotations revealed a general shift in fungal gene expression in which genes related to xenobiotic degradation, toxin and antioxidant production, energy, carbohydrate and lipid metabolism and hyphal rearrangements were subjected to transcriptional regulation.ConclusionsThis RNA-seq profiling generated a novel dataset describing the functional response of the phytopathogen R. solani AG3 to the plant-associated Serratia bacteria S4 and AS13. Most genes were regulated in the same way in the presence of both bacterial isolates, but there were also some strain-specific responses. The findings in this study will be beneficial for further research on biological control and in depth exploration of bacterial-fungal interactions in the rhizosphere.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1758-z) contains supplementary material, which is available to authorized users.

Selection of bioprotective cultures for preventing cold-smoked salmon spoilage

Biopreservation is a natural technology of food preservation, which consists of inoculating food with microorganisms selected for their antibacterial properties. The objective of this study was to select lactic acid bacteria (LAB) to improve the quality of cold-smoked salmon (CSS). In this work, different strains representative of the 4 dominant species, identified in a previous study by pyrosequencing the 16S rRNA gene, were isolated and their spoiling potential in CSS blocks, sterilized by ionization, was assessed by twelve trained panelists along the vacuum storage at 8°C. Photobacterium phosphoreum, Brochothrix thermosphacta and Serratia proteamaculans released strong off-odors whereas the spoiling potential of Carnobacterium divergens was weaker. The spoiling capacity of Lactococcus piscium EU2241, Leuconostoc gelidum EU2247, Lactobacillus sakei EU2885, Staphylococcus equorum S030674 and 4 commercial starters was tested by the same method and 2 strains were eliminated due to off-odor production. The effect of the 6 selected LAB against the 4 specific spoiling organisms (SSOs) selected was tested by challenge tests in sterile CSS blocks. The protective effect of the LAB differed from one SSO to another and no correlation could be established between the sensory improvement, SSO inhibition, and the implantation or acidification of protective cultures (PCs). All the PCs except L. piscium reduced the off-odors released by P. phosphoreum although some of them had no effect on its growth. S. equorum, which did not grow in CSS, favored the implantation of P. phosphoreum but prevented its off-odor formation. L. piscium was the only strain that prevented the spoilage of B. thermosphacta and S. proteamaculans although it did not grow very well and did not acidify the product. L. gelidum EU2247 inhibited the growth of these 2 SSOs and lowered the pH but had no effect on the sensory quality. Finally, L. piscium was tested in 2 naturally contaminated products, with a positive effect on 1 batch. This effect was not correlated with the microbial ecosystem as determined by acultural and cultural techniques. Based on these results, the selection strategy is discussed.

Catalytic efficiency of chitinase-D on insoluble chitinous substrates was improved by fusing auxiliary domains.

Chitin is an abundant renewable polysaccharide, next only to cellulose. Chitinases are important for effective utilization of this biopolymer. Chitinase D from Serratia proteamaculans (SpChiD) is a single domain chitinase with both hydrolytic and transglycosylation (TG) activities. SpChiD had less of hydrolytic activity on insoluble polymeric chitin substrates due to the absence of auxiliary binding domains. We improved catalytic efficiency of SpChiD in degradation of insoluble chitin substrates by fusing with auxiliary domains like polycystic kidney disease (PKD) domain and chitin binding protein 21 (CBP21). Of the six different SpChiD fusion chimeras, two C-terminal fusions viz. ChiD+PKD and ChiD+CBP resulted in improved hydrolytic activity on α- and β-chitin, respectively. Time-course degradation of colloidal chitin also confirmed that these two C-terminal SpChiD fusion chimeras were more active than other chimeras. More TG products were produced for a longer duration by the fusion chimeras ChiD+PKD and PKD+ChiD+CBP.

Virulence factors contributing to invasive activities of Serratia grimesii and Serratia proteamaculans.

Previously, we have shown that facultative pathogens Serratia grimesii and Serratia proteamaculans are capable to invade eukaryotic cells provided that they synthesize intracellular metalloprotease grimelysin or protealysin, respectively (Bozhokina et al. in Cell Biol Int 35(2):111-118, 2011). Noninvasive Escherichia coli transformed with grimelysin or protealysin gene became invasive, indicating that the protease is a virulence factor. Here we elucidated involvement of other virulence factors in the invasion of S. grimesii and S. proteamaculans. Under similar experimental conditions, the amount of S. proteamaculans internalized within human carcinoma HeLa cells was fivefold higher than that of S. grimesii. In accord with this, in S. proteamaculans, high activities of pore-forming hemolysin ShlA and extracellular metalloprotease serralysin were detected. In S. grimesii, activity of toxin ShlA was not detected, and the serralysin activity of the bacterial growth medium was very low. We also show that iron depletion strongly enhanced invasive activity of S. proteamaculans, increasing activities of hemolysin ShlA and serralysin, but did not affect S. grimesii properties. These results show that the invasive activity of S. proteamaculans is maintained, along with protealysin, by hemolysin and serralysin. On the other hand, grimelysin is so far the only known invasion factor of S. grimesii.

一株解烃菌TY12的分离及其特性研究

本文以南充市炼油厂附近被石油污染获取的土壤作为研究材料，筛选分离获得一株对石油烃有降解性的疏水性烷烃降解菌TY12。经过16S rDNA的克隆测序和系统发育分析，初步确定该菌株为变形斑沙雷氏菌(Serratia proteamaculans)，并从形态学、生理生化特性对发现的菌株TY12进行了进一步的研究和分析。同时用微生物粘着碳烃化合物法(MATH)研究得到TY12菌株的表面疏水性为52.2%，且表面疏水性会随着培养时间的增加，不断的减小。该菌能在以正十二烷为唯一碳源的无机盐培养基中生长，当在含有1%(W/V)正十二烷的无机盐培养基中接入1%种子液，28℃、摇床转速180 r/min的条件下震荡培养4 d，正十二烷降解率可达66.1%。其研究成果为石油污染土壤的生物修复提供了一定的依据。 A strain of hydrophobic hydrocarbon degrading bacteria TY12 was screened out of petroleum contaminated soils from Nanchong oil refinery. Through 16S rDNA basic sequence detection and phylogenetic analysis, TY12 was identified as Serratia proteamaculans, and then according to morphological, physiology and biochemistry characteristics, this paper has a deep study on TY12. This paper studied on the cell-surface hydrophobicity of TY12 by microorganism adhering to hy-drocarbon (MATH), the results showed that the cell-surface hydrophobicity of TY12 was 52.2%, and the cell-surface hydrophobicity changed with the variation of incubation time. The n-dodecane degradation was 66.1% after TY12 was growing on in hydrocarbon degradation medium with 1% (W/V) of n-docosane at 28˚C and 180 r/min for 4 d. It had potential in bioremediation of oil con-taminated environment.

Discovery of a bifunctional acyltransferase responsible for ornithine lipid synthesis in Serratia proteamaculans

Ornithine lipids (OLs) are phosphorus-free membrane lipids that can be formed by many bacteria but that are absent from archaea and eukaryotes. A function for OLs in stress conditions and in host-bacteria interactions has been shown in some bacteria. Some bacterial species have been described that can form OLs, but lack the known genes (olsBA) involved in its biosynthesis, which implied the existence of a second pathway. Here we describe the bifunctional protein OlsF from Serratia proteamaculans involved in OL formation. Expression of OlsF and its homologue from Flavobacterium johnsoniae in Escherichia coli causes OL formation. Deletion of OlsF in S. proteamaculans caused the absence of OL formation. Homologues of OlsF are widely distributed among γ-, δ- and ε-Proteobacteria and in the Cytophaga-Flavobacterium-Bacteroidetes group of bacteria, including several well-studied pathogens for which the presence of OLs has not been suspected, such as for example Vibrio cholerae and Klebsiella pneumonia. Using genomic data, we predict that about 50% of bacterial species can form OLs.

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.

Mutagenesis and molecular dynamics simulations revealed the chitooligosaccharide entry and exit points for chitinase D from Serratia proteamaculans

BackgroundTransglycosylation (TG) activity is a property of glycosyl hydrolases (GHs) with which new glycosidic bonds are introduced between donor and acceptor sugar molecules. This special property of the GHs has potential to generate longer chain chitooligosaccharides (CHOS) that show elicitor activity in plants. We hypothesize that TG activity could be improved by retaining the substrate for a longer duration in the catalytic site. MethodsFour variants of chitinase D from Serratia proteamaculans (SpChiD) i.e. G119S, G119W, W120A and G201W were analyzed in detail for improved TG activity using high performance liquid chromatography (HPLC) and high resolution mass spectrometry (HRMS). The results were strongly supported by 50ns molecular dynamics (MD) simulations and estimated solvated interaction energies (SIE). ResultsThe mutant G119W lost much of both hydrolytic and TG activities, while the mutant G201W displayed increased TG. The trajectory of MD simulations of the mutant G119W showed that the indole rings of two adjacent Trp residues create a major hindrance for the DP4 movement towards the catalytic center. Increased van der Waals (vdW) and coulombic interactions between DP4 substrate and the Trp-201 resulted in enhanced TG activity with the mutant G201W. The average number of hydrogen bonds observed for the DP4 substrate was increased for the mutants G119W and G201W compared to SpChiD. ConclusionThe increase in TG activity could be due to partial blocking of product exit of SpChiD. General significanceThis new approach can be used for generating mutants of GHs with improved TG activity to produce longer chain oligosaccharides.

High-level production of Serratia proteamaculans metalloprotease using a recombinant ABC protein exporter-mediated secretion system in Pseudomonas fluorescens

Serratia proteamaculans metalloprotease (SPP) was successfully secreted by a heterologous ABC protein exporter, the Pseudomonas fluorescens TliDEF, in recombinant host strains. Escherichia coli and P. fluorescens cells containing the SPP-encoding gene showed the extracellular protease activity only when the TliDEF-encoding gene cluster was coexpressed. Recombinant P. fluorescens produced an approximately 34.8-fold higher amount of extracellular SPP than did E. coli. The use of a more nutrient-rich medium and controlled dissolved oxygen conditions was effective in increasing SPP secretion in P. fluorescens batch fermentation (an 8.7-fold increase from 41.8U/mL to 365.2U/mL). Therefore, SPP, which could not be secreted without an ABC protein exporter, was produced in large quantities by applying the heterologous TliDEF exporter in P. fluorescens. The results also suggest that the use of the ABC protein exporter in P. fluorescens could be an efficient production platform for an industrially promising type I secretion pathway-dependent enzyme.

The spoilage characteristics of Brochothrix thermosphacta and two psychrotolerant Enterobacteriacae in vacuum packed lamb and the comparison between high and low pH cuts

The spoilage potential of Brochothrix thermosphacta, Serratia proteamaculans and Rahnella aquatilis was investigated in vacuum packaged high (5.9 to 6.4) and low (5.4 to 5.8) pH lamb. Vacuum packaged fore shank (m. extensor carpi radialis) and striploins (m. longissimus dorsi) (n=306) inoculated with ~100CFU of individual bacteria were stored for twelve weeks at temperatures −1.5, 0, 2 and 7°C. Spoilage characteristics and bacterial numbers were recorded and analysed in comparison to un-inoculated control samples. All three bacterial species were shown to grow in vacuum packaged lamb of pH values between 5.4 and 6.4, when stored at chilled temperatures (−1.5 to 7°C) for up to 84days. B. thermosphacta and S. proteamaculans caused spoilage to the meat under these conditions whilst R. aquatilis spoiled high pH meat at 7°C. These results go against previous beef models stipulating that Brochothrix and Enterobacteriacae species cannot grow on or cause spoilage of low pH meat in the absence of oxygen.

Unusual metallo-<i>β</i>-lactamases may constitute a new subgroup in this family of enzymes

Metallo-β-lactamases (MBLs) are a family of Zn2+-dependent enzymes that have contributed strongly to the emergence and spread of antibiotic resistance. Novel members as well as variants of existing members of this family are discovered continuously, compounding their threat to global health care. MBLs are divided into three subgroups, i.e. B1, B2 and B3. The recent discovery of an unusual MBL from Serratia proteamaculans (SPR-1) suggests the presence of an additional subgroup, i.e. B4. A database search reveals that SPR-1 has only one homologue from Cronobacter sakazakii, CSA-1.These two MBLs have a unique active site and may employ a mechanism distinct from other MBLs, but reminiscent of some organophosphate-degrading hydrolases.

Hexavalent chromium removal by a novel Serratia proteamaculans isolated from the bank of Sebou River (Morocco)

The novel Serratia proteamaculans isolated from a chromium-contaminated site was tolerant to a concentration of 500 mg Cr(VI)/l. The optimum pH and temperature for reduction of Cr(VI) by S. proteamaculans were found to be 7.0 and 30 °C, respectively. The Cr(VI) reduction rate decreased with the increase in Cr(VI) concentration from 100 to 400 mg/l, suggesting the enzymatic chromium reduction. Resting and permeabilised cell assays provided the better evidence that chromate reduction in S. proteamaculans is enzymatic. Reduction by cell-free filtrate shows no extracellular chromate-reducing activity, revealing that this activity may be associated to membrane fraction and/or cytosolic fraction. Assays conducted with cytosolic and particulate fraction of S. proteamaculans confirmed the role of membrane-bound proteins in Cr(VI) reduction. Furthermore, chromium reduced by heat-treated cells suggests that membrane-associated chromate reductase activity of S. proteamaculans is preceded by its adsorption on the cell surface.

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 °С 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.