Cloning and expression of recombinant arazyme with anti-inflammatory and anti-breast cancer potential.

Arazyme is an extracellular metalloprotease which is secreted by a Gram-negative symbiotic bacterium called Serratia proteomaculans. There are limited studies on various biological activities of arazyme. This preliminary study was designed to investigate the anti-cancer and anti-inflammatory capacities of recombinant arazyme (rAra) in vitro and in vivo. Arazyme gene, araA was cloned and expressed in E. coli BL21 (DE3) using pET-28a as a vector. Nickel column purification was used to obtain pure rAra. SDS-PAGE and protein assay were used to identify the product and to measure protein content, respectively. Skimmed milk test and casein assay were carried out to assess protease activity. MCF7 cells as a breast cancer cell model were exposed to different concentrations of rAra to study anti-breast cancer potentials using MTT assay. The anti-inflammatory property of rAra was investigated using a murine air-pouch model. PCR and SDS-PAGE data showed that cloning and expression of rAra was successful and the enzyme of interest was observed at 52 KDa. Protein assay indicated that 1 mg/ml of rAra was obtained through purification. A clear zone around the enzyme on skimmed milk agar confirmed the proteolytic activity of rAra and the enzymatic activity was 320 U/mg protein in the casein assay. Cytotoxic effects of rAra reported as IC50 were 16.2 µg/ml and 13.2 mg/ml after 24 h and 48 h, respectively. In the air-pouch model, both the neutrophil count and myeloperoxidase activity, which are measures of inflammation, were significantly reduced. The results showed that rAra can be used in future mechanistic studies and R&D activities in the pharmaceutical industry to investigate the safety and efficacy of the recombinant arazyme.

Modular Halogenation, α-Hydroxylation, and Acylation by a Remarkably Versatile Polyketide Synthase.

Bacterial multimodular polyketide synthases (PKSs) are large enzymatic assembly lines that synthesize many bioactive natural products of therapeutic relevance. While PKS catalysis is mostly based on fatty acid biosynthetic principles, polyketides can be further diversified by post-PKS enzymes. Here, we characterized a remarkably versatile trans-acyltransferase (trans-AT) PKS from Serratia that builds structurally complex macrolides via more than ten functionally distinct PKS modules. In the oocydin PKS, we identified a new oxygenation module that α-hydroxylates polyketide intermediates, a halogenating module catalyzing backbone γ-chlorination, and modular O-acetylation by a thioesterase-like domain. These results from a single biosynthetic assembly line highlight the expansive biochemical repertoire of trans-AT PKSs and provide diverse modular tools for engineered biosynthesis from a close relative of E. coli.

Serratia sp., an endophyte of Mimosa pudica nodules with nematicidal, antifungal activity and growth-promoting characteristics.

In the present study, the nematicidal activity of an isolated strain of Mimosa pudica nodules was evaluated against the Nacobbus aberrans (J2) phytonymatodes with a mortality of 88.8%, while against the gastrointestinal nematode Haemonchus contortus (L3) and free-living Panagrellus redivivus was 100%. The ability to inhibit the growth of phytopathogenic fungi Fusarium sp., and Alternaria solani, as well as the oomycete Phytophthora capsici, this antifungal activity may be related to the ability to produce cellulases, siderophores and chitinases by this bacterial strain. Another important finding was the detection of plant growth promoter characteristics, such as auxin production and phosphate solubilization. The strain identified by sequences of the 16S and rpoB genes as Serratia sp. is genetically related to Serratia marcescens and Serratia nematodiphila. The promoter activity of plant growth, antifungal and nematicide of the Serratia sp. strain makes it an alternative for the biocontrol of fungi and nematodes that affect both the livestock and agricultural sectors, likewise, candidate as a growth-promoting bacterium.

Crystal Structure of a Phospholipase D from the Plant-Associated Bacteria Serratia plymuthica Strain AS9 Reveals a Unique Arrangement of Catalytic Pocket.

Phospholipases D (PLDs) play important roles in different organisms and in vitro phospholipid modifications, which attract strong interests for investigation. However, the lack of PLD structural information has seriously hampered both the understanding of their structure-function relationships and the structure-based bioengineering of this enzyme. Herein, we presented the crystal structure of a PLD from the plant-associated bacteria Serratia plymuthica strain AS9 (SpPLD) at a resolution of 1.79 Å. Two classical HxKxxxxD (HKD) motifs were found in SpPLD and have shown high structural consistence with several PLDs in the same family. While comparing the structure of SpPLD with the previous resolved PLDs from the same family, several unique conformations on the C-terminus of the HKD motif were demonstrated to participate in the arrangement of the catalytic pocket of SpPLD. In SpPLD, an extented loop conformation between ß9 and α9 (aa228-246) was found. Moreover, electrostatic surface potential showed that this loop region in SpPLD was positively charged while the corresponding loops in the two Streptomyces originated PLDs (PDB ID: 1F0I, 2ZE4/2ZE9) were neutral. The shortened loop between α10 and α11 (aa272-275) made the SpPLD unable to form the gate-like structure which existed specically in the two Streptomyces originated PLDs (PDB ID: 1F0I, 2ZE4/2ZE9) and functioned to stabilize the substrates. In contrast, the shortened loop conformation at this corresponding segment was more alike to several nucleases (Nuc, Zuc, mZuc, NucT) within the same family. Moreover, the loop composition between ß11 and ß12 was also different from the two Streptomyces originated PLDs (PDB ID: 1F0I, 2ZE4/2ZE9), which formed the entrance of the catalytic pocket and were closely related to substrate recognition. So far, SpPLD was the only structurally characterized PLD enzyme from Serratia. The structural information derived here not only helps for the understanding of the biological function of this enzyme in plant protection, but also helps for the understanding of the rational design of the mutant, with potential application in phospholipid modification.

Modulation of the Biocatalytic Properties of a Novel Lipase from Psychrophilic Serratia sp. (USBA-GBX-513) by Different Immobilization Strategies.

In this work, the effect of different immobilization procedures on the properties of a lipase obtained from the extremophilic microorganism Serratia sp. USBA-GBX-513, which was isolated from Paramo soils of Los Nevados National Natural Park (Colombia), is reported. Different Shepharose beads were used: octyl-(OC), octyl-glyoxyl-(OC-GLX), cyanogen bromide (BrCN)-, and Q-Sepharose. The performance of the different immobilized extremophile lipase from Serratia (ESL) was compared with that of the lipase B from Candida antarctica (CALB). In all immobilization tests, hyperactivation of ESL was observed. The highest hyperactivation (10.3) was obtained by immobilization on the OC support. Subsequently, the thermal stability at pH 5, 7, and 9 and the stability in the presence of 50% (v/v) acetonitrile, 50% dioxane, and 50% tetrahydrofuran solvents at pH 7 and 40 °C were evaluated. ESL immobilized on octyl-Sepharose was the most stable biocatalyst at 90 °C and pH 9, while the most stable preparation at pH 5 was ESL immobilized on OC-GLX-Sepharose supports. Finally, in the presence of 50% (v/v) tetrahydrofuran (THF) or dioxane at 40 °C, ESL immobilized on OC-Sepharose was the most stable biocatalyst, while the immobilized preparation of ESL on Q-Sepharose was the most stable one in 40% (v/v) acetonitrile.

Chemoenzymatic Synthesis of Chito-oligosaccharides with Alternating N-d-Acetylglucosamine and d-Glucosamine.

Chito-oligosaccharides (CHOS) are homo- or hetero-oligomers of N-acetylglucosamine (GlcNAc, A) and d-glucosamine (GlcN, D). Production of well-defined CHOS-mixtures, or even pure CHOS, with specific lengths and sugar compositions, is of great interest since these oligosaccharides have interesting bioactivities. While direct chemical synthesis of CHOS is not straightforward, chemo-enzymatic approaches have shown some promise. We have used engineered glycoside hydrolases to catalyze oligomerization of activated DA building blocks through transglycosylation reactions. The building blocks were generated from readily available (GlcNAc)2-para-nitrophenol through deacetylation of the nonreducing end sugar with a recombinantly expressed deacetylase from Aspergillus niger (AnCDA9). This approach, using a previously described hyper-transglycosylating variant of ChiA from Serratia marcescens (SmChiA) and a newly generated transglycosylating variant of Chitinase D from Serratia proteamaculans (SpChiD), led to production of CHOS containing up to ten alternating D and A units [(DA)2, (DA)3, (DA)4, and (DA)5]. The most abundant compounds were purified and characterized. Finally, we demonstrate that (DA)3 generated in this study may serve as a specific inhibitor of the human chitotriosidase. Inhibition of this enzyme has been suggested as a therapeutic strategy against systemic sclerosis.

Engineering a Highly Active Sucrose Isomerase for Enhanced Product Specificity by Using a "Battleship" Strategy.

The sucrose isomerase SmuA from Serratia plymuthica efficiently catalyses the isomerisation of sucrose into isomaltulose, an artificial sweetener used in the food industry. However, the formation of a hygroscopic by-product, trehalulose, necessitates additional separation to obtain a crystalline product. Therefore, we have improved the product specificity of SmuA by first introducing a few exploratory amino acid exchanges around the active site and investigating their influence. Then, we devised a second set of mutations, either at promising positions from the preceding cycle, but with a different side chain, or at alternative positions in the vicinity. After seven iterative cycles involving just 55 point mutations, we obtained the triple mutant Y219L/D398G/V465E which showed 2.3 times less trehalulose production but still had high catalytic efficiency (kcat /KM =11.8 mM-1 s-1 ). Not only does this mutant SmuA appear attractive as an industrial biocatalyst, but our semirational protein-engineering strategy, which resembles the battleship board game, should be of interest for other challenging enzyme optimization endeavours.

Substrate Flexibility of the Flavin-Dependent Dihydropyrrole Oxidases PigB and HapB Involved in Antibiotic Prodigiosin Biosynthesis.

In the biosynthesis of the tripyrrolic pigment prodigiosin, PigB is a predicted flavin-dependent oxidase responsible for the formation of 2-methyl-3-amylpyrrole (MAP) from a dihydropyrrole. To prove which dihydropyrrole is the true intermediate, both possibilities, 5-methyl-4-pentyl-3,4-dihydro-2H-pyrrole (5 a, resulting from transamination of the aldehyde of 3-acetyloctanal) and 2-methyl-3-pentyl-3,4-dihydro-2H-pyrrole (6, resulting from transamination of the ketone), were synthesised. Only 5 a restored pigment production in a strain of Serratia sp. ATCC 39006 blocked earlier in MAP biosynthesis. PigB is membrane-associated and inactive when its transmembrane domain was deleted, but HapB, its homologue in Hahella chejuensis, lacks the transmembrane domain and is active in solution. Two colourimetric assays for PigB and HapB were developed, and the HapB-catalysed reaction was kinetically characterised. Ten analogues of 5 a were synthesised, varying in the C2 and C3 side chains, and tested as substrates of HapB in vitro and for restoration of pigment production in Serratia ΔpigD in vivo. All lengths of side chain tested at C3 were accepted, but only short side chains at C2 were accepted. The knowledge that 5 a is an intermediate in prodigiosin biosynthesis and the ease of synthesis of analogues of 5 a makes a range of prodigiosin analogues readily available by mutasynthesis.

Production optimization, characterization, and covalent immobilization of a thermophilic Serratia rubidaea lipase isolated from an Algerian oil waste.

A new thermophilic non-induced lipase producer named Serratia rubidaea strain Nehal-mou was isolated from oil waste in Tissemsilat, Algeria. The most influential lipase production parameters were screened by the Plackett-Burman design for enhancing enzyme yield. An optimum condition of a 1.5% of glucose, a 0.01% of potassium, and a 0.025% of manganese contents resulted in a 41.13 U/mL. This yield was 6.29 times higher than the one achieved before the application of the Box-Behnken Design. Lipase activity showed a high organic solvent tolerance following its exposure to hexane, ethanol, methanol, and acetone. Lipase was also perfectly stable in the presence of 10 mM Fe2+, K+, and Na+ ions with more than 75% of the retaining activity. The enzyme half-life times were 22 h, 90 min, and 25 min at 50, 60, and 70 °C respectively. Polyvinyl alcohol (PVA)/boric acid/Starch/CaCO3 were utilized as a carrier for lipase covalent immobilization in order to be used efficiently. The Scanning Electron Microscopy (SEM) Technique and the Fourier Transform Infrared Spectroscopy (FTIR) Method confirmed the covalent bonding success and the excellent carrier characteristics. Thus, the immobilization yield reached 73.5% and the optimum temperature was shifted from 40 to 65 °C. The immobilized lipase kept 80% of its total activity after 10 cycles and had 3 and 3.2-fold half-lives at 70, and 80 °C respectively compared to the free enzyme.

Purification and characterization of the thermostable protease produced by Serratia grimesii isolated from channel catfish.

BACKGROUND: Microbial spoilage of fishery products accounts for significant financial losses, yearly on a global scale. Psychrotrophic spoilage bacteria often secrete extracellular enzymes to break down surrounding fish tissue, rendering the product unsuitable for human consumption. For a better understanding of bacterial spoilage due to enzymatic digestion of fish products, proteases in Serratia grimesii isolated from North American catfish fillets (Ictalurus punctatus) were investigated. RESULTS: Mass spectrometric evidence demonstrated that S. grimesii secretes two distinct extracellular proteases and one lipase. Protease secretion displayed broad thermostability in the 30-90 °C range. The major protease-secretion (O-1) was most active under alkaline conditions and utilized manganese as a co-factor. Organic solvents significantly disrupted the efficacy of S. grimesii extracellular enzymes and, in a series of bactericidal detergents, protease activity was highest when treated with Triton X-100. Ethylenediaminetetraacetic acid (EDTA) and phenylmethylsulfonyl fluoride (PMSF) significantly inhibited the enzyme activity, while protease was moderately stable under freeze-thaw and refrigerated storage. CONCLUSION: The influence of fish spoilage-related enzymes, depending on various factors, is discussed in this paper. This study will provide new insight into enzymatic spoilage and its control, which can be exploited to enhance food safety and the shelf-life of fishery products worldwide. © 2018 Society of Chemical Industry.

Key Residues Affecting Transglycosylation Activity in Family 18 Chitinases: Insights into Donor and Acceptor Subsites.

Understanding features that determine transglycosylation (TG) activity in glycoside hydrolases is important because it would allow the construction of enzymes that can catalyze controlled synthesis of oligosaccharides. To increase TG activity in two family 18 chitinases, chitinase D from Serratia proteamaculans ( SpChiD) and chitinase A from Serratia marcescens ( SmChiA), we have mutated residues important for stabilizing the reaction intermediate and substrate binding in both donor and acceptor sites. To help mutant design, the crystal structure of the inactive SpChiD-E153Q mutant in complex with chitobiose was determined. We identified three mutations with a beneficial effect on TG activity: Y28A (affecting the -1 subsite and the intermediate), Y222A (affecting the intermediate), and Y226W (affecting the +2 subsite). Furthermore, exchange of D151, the middle residue in the catalytically important DXDXE motif, to asparagine reduced hydrolytic activity ≤99% with a concomitant increase in apparent TG activity. The combination of mutations yielded even higher degrees of TG activity. Reactions with the best mutant, SpChiD-D151N/Y226W/Y222A, led to rapid accumulation of high levels of TG products that remained stable over time. Importantly, the introduction of analogous mutations at the same positions in SmChiA (Y163A equal to Y28A and Y390F similar to Y222A) had similar effects on TG efficiency. Thus, the combination of the decreasing hydrolytic power, subsite affinity, and stability of intermediate states provides a powerful, general strategy for creating hypertransglycosylating mutants of retaining glycoside hydrolases.

Sodorifen Biosynthesis in the Rhizobacterium Serratia plymuthica Involves Methylation and Cyclization of MEP-Derived Farnesyl Pyrophosphate by a SAM-Dependent C-Methyltransferase.

The rhizobacterium Serratia plymuthica 4Rx13 releases a unique polymethylated hydrocarbon (C16H26) with a bicyclo[3.2.1]octadiene skeleton called sodorifen. Sodorifen production depends on a gene cluster carrying a C-methyltransferase and a terpene cyclase along with two enzymes of the 2- C-methyl-d-erythritol 4-phosphate (MEP) pathway of isoprenoid biosynthesis. Comparative analysis of wild-type and mutant volatile organic compound profiles revealed a C-methyltransferase-dependent C16 alcohol called pre-sodorifen, the production of which is upregulated in the terpene cyclase mutant. The monocyclic structure of this putative intermediate in sodorifen biosynthesis was identified by NMR spectroscopy. In vitro assays with the heterologously expressed S. plymuthica C-methyltransferase and terpene cyclase demonstrated that these enzymes act sequentially to convert farnesyl pyrophosphate (FPP) into sodorifen via a pre-sodorifen pyrophosphate intermediate, indicating that the S-adenosyl methionine (SAM)-dependent C-methyltransferase from S. plymuthica exhibits unprecedented cyclase activity. In vivo incorporation experiments with 13C-labeled succinate, l-alanine, and l-methionine confirmed a MEP pathway to FPP via the canonical glyceraldehyde-3-phosphate and pyruvate, as well as its SAM-dependent methylation in pre-sodorifen and sodorifen biosynthesis. 13C{1H} NMR spectroscopy facilitated the localization of 13C labels and provided detailed insights into the biosynthetic pathway from FPP via pre-sodorifen pyrophosphate to sodorifen.

Monoacylglycerol and diacylglycerol production by hydrolysis of refined vegetable oil by-products using an immobilized lipase from Serratia sp. W3.

Unlike the synthetic surfactants, mono- and diacylglycerols have the advantage to be biodegradable and non-toxic. In the present work, the hydrolysis of lipid fraction by-products of refined vegetable oils was performed by Serratia sp. W3 lipase immobilized on CaCO3 by combined adsorption and precipitation. This support was selected out of four carriers as it exhibited the finest activity support (950 U/g) and the most satisfactory behavior at use. The immobilized preparation with CaCO3 was stable and active in the whole range of pH (4 to 9) and temperature (37 to 55°C), yielding a 75% degree of hydrolysis at optimal environmental conditions of pH 8.5 and temperature 55°C. Thin-layer chromatography, gas chromatography, and liquid chromatography methods were evaluated to determine the analytical characterization of hydrolysis products. For monoacylglycerols and diacylglycerol fractions identified in the samples, a novel approach by liquid chromatography method was employed, through a homemade linear retention index database and a dedicated software. The adopted approach allowed the use of basic instrumentation set-ups, without the need of sophisticated detectors, such as mass spectrometers. Thus, it could be an effective alternative to produce emulsifiers from cheap vegetable oils.

The expression of an exogenous ACC deaminase by the endophyte Serratia grimesii BXF1 promotes the early nodulation and growth of common bean.

Ethylene acts as an inhibitor of the nodulation process of leguminous plants. However, some bacteria can decrease deleterious ethylene levels by the action of the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase which degrades ACC, the ethylene precursor in all higher plants. Co-inoculation of rhizobia with endophytes enhances the rhizobial symbiotic efficiency with legumes, improving both nodulation and nitrogen fixation. However, not much is understood about the mechanisms employed by these endophytic bacteria. In this regard, the role of ACC deaminase from endophytic strains in assisting rhizobia in this process has yet to be confirmed. In this study, the role of ACC deaminase in an endophyte's ability to increase Rhizobium tropici nodulation of common bean was evaluated. To assess the effect of ACC deaminase in an endophyte's ability to promote rhizobial nodulation, the endophyte Serratia grimesii BXF1, which does not encode ACC deaminase, was transformed with an exogenous acdS gene. The results obtained indicate that the ACC deaminase-overexpressing transformant strain increased common bean growth, and enhanced the nodulation abilities of R. tropici CIAT899, in both cases compared to the wild-type non-transformed strain. Furthermore, plant inoculation with the ACC deaminase-overproducing strain led to an increased level of plant protection against a seed-borne pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: In this work, we studied the effect of ACC deaminase production by the bacterial endophyte Serratia grimesi BXF1, and its impact on the nodulation process of common bean. The results obtained indicate that ACC deaminase is an asset to the synergetic interaction between rhizobia and the endophyte, positively contributing to the overall legume-rhizobia symbiosis by regulating inhibitory ethylene levels that might otherwise inhibit nodulation and overall plant growth. The use of rhizobia together with an ACC deaminase-producing endophyte is, therefore, an important strategy for the development of new bacterial inoculants with increased performance.

A Polyketide Synthase Component for Oxygen Insertion into Polyketide Backbones.

Enzymatic core components from trans-acyltransferase polyketide synthases (trans-AT PKSs) catalyze exceptionally diverse biosynthetic transformations to generate structurally complex bioactive compounds. Here we focus on a group of oxygenases identified in various trans-AT PKS pathways, including those for pederin, oocydins, and toblerols. Using the oocydin pathway homologue (OocK) from Serratia plymuthica 4Rx13 and N-acetylcysteamine (SNAC) thioesters as test surrogates for acyl carrier protein (ACP)-tethered intermediates, we show that the enzyme inserts oxygen into ß-ketoacyl moieties to yield malonyl ester SNAC products. Based on these data and the identification of a non-hydrolyzed oocydin congener with retained ester moiety, we propose a unified biosynthetic pathway of oocydins, haterumalides, and biselides. By providing access to internal ester, carboxylate pseudostarter, and terminal hydroxyl functions, oxygen insertion into polyketide backbones greatly expands the biosynthetic scope of PKSs.

Inactivation of the Major Hemolysin Gene Influences Expression of the Nonribosomal Peptide Synthetase Gene swrA in the Insect Pathogen Serratia sp. Strain SCBI.

Hemolysins are important virulence factors for many bacterial pathogens, including Serratia marcescens The role of the major hemolysin gene in the insect pathogen Serratia sp. strain SCBI was investigated using both forward and reverse-genetics approaches. Introduction of the major hemolysin gene into Escherichia coli resulted in a gain of both virulence and hemolytic activity. Inactivation of this hemolysin in Serratia sp. SCBI resulted in a loss of hemolysis but did not attenuate insecticidal activity. Unexpectedly, inactivation of the hemolysin gene in Serratia sp. SCBI resulted in significantly increased motility and increased antimicrobial activity. Reverse transcription-quantitative PCR (qRT-PCR) analysis of mutants with a disrupted hemolysin gene showed a dramatic increase in mRNA levels of a nonribosomal peptide synthetase gene, swrA, which produces the surfactant serrawettin W2. Mutation of the swrA gene in Serratia sp. SCBI resulted in highly varied antibiotic activity, motility, virulence, and hemolysis phenotypes that were dependent on the site of disruption within this 17.75-kb gene. When introduced into E. coli, swrA increases rates of motility and confers antimicrobial activity. While it is unclear how inactivation of the major hemolysin gene influences the expression of swrA, these results suggest that swrA plays an important role in motility and antimicrobial activity in Serratia sp. SCBI.IMPORTANCE The opportunistic Gram-negative bacteria of the genus Serratia are widespread in the environment and can cause human illness. A comparative genomics analysis between Serratia marcescens and a new Serratia species from South Africa, termed Serratia sp. strain SCBI, shows that these two organisms are closely related but differ in pathogenesis. S. marcescens kills Caenorhabditis nematodes, while Serratia sp. SCBI is not harmful and forms a beneficial association with them. This distinction presented the opportunity to investigate potential differences in regulation of common virulence mechanisms between these two species. With the emergence of antibiotic-resistant microorganisms, there is a widespread need to understand the regulation of pathogenesis. The significance of this study is the presentation of evidence for cross-pathway regulation of virulence factors and how the elimination of one mechanism may be compensated for by the upregulation of others.

Concentration and Variety of Carbapenemase Producers in Recreational Coastal Waters Showing Distinct Levels of Pollution.

Carbapenemase-producing bacteria cause difficult-to-treat infections related to increased mortality in health care settings. Their occurrence has been reported in raw sewage, sewage-impacted rivers, and polluted coastal waters, which may indicate their spread to the community. We assessed the variety and concentration of carbapenemase producers in coastal waters with distinct pollution levels for 1 year. We describe various bacterial species producing distinct carbapenemases not only in unsuitable waters but also in waters considered suitable for primary contact.

Chromosome-Encoded Broad-Spectrum Ambler Class A ß-Lactamase RUB-1 from Serratia rubidaea.

Whole-genome sequencing of Serratia rubidaea CIP 103234T revealed a chromosomally located Ambler class A ß-lactamase gene. The gene was cloned, and the ß-lactamase, RUB-1, was characterized. RUB-1 displayed 74% and 73% amino acid sequence identity with the GIL-1 and TEM-1 penicillinases, respectively, and its substrate profile was similar to that of the latter ß-lactamases. Analysis by 5' rapid amplification of cDNA ends revealed promoter sequences highly divergent from the Escherichia coli σ70 consensus sequence. This work further illustrates the heterogeneity of ß-lactamases among Serratia spp.

Crystal structures and inhibitor binding properties of plant class V chitinases: the cycad enzyme exhibits unique structural and functional features.

A class V (glycoside hydrolase family 18) chitinase from the cycad Cycas revoluta (CrChiA) is a plant chitinase that has been reported to possess efficient transglycosylation (TG) activity. We solved the crystal structure of CrChiA, and compared it with those of class V chitinases from Nicotiana tabacum (NtChiV) and Arabidopsis thaliana (AtChiC), which do not efficiently catalyze the TG reaction. All three chitinases had a similar (α/ß)8 barrel fold with an (α + ß) insertion domain. In the acceptor binding site (+1, +2 and +3) of CrChiA, the Trp168 side chain was found to stack face-to-face with the +3 sugar. However, this interaction was not found in the identical regions of NtChiV and AtChiC. In the DxDxE motif, which is essential for catalysis, the carboxyl group of the middle Asp (Asp117) was always oriented toward the catalytic acid Glu119 in CrChiA, whereas the corresponding Asp in NtChiV and AtChiC was oriented toward the first Asp. These structural features of CrChiA appear to be responsible for the efficient TG activity. When binding of the inhibitor allosamidin was evaluated using isothermal titration calorimetry, the changes in binding free energy of the three chitinases were found to be similar to each other, i.e. between -9.5 and -9.8 kcal mol(-1) . However, solvation and conformational entropy changes in CrChiA were markedly different from those in NtChiV and AtChiC, but similar to those of chitinase A from Serratia marcescens (SmChiA), which also exhibits significant TG activity. These results provide insight into the molecular mechanism underlying the TG reaction and the molecular evolution from bacterial chitinases to plant class V chitinases.

Biosynthesis of the acetyl-CoA carboxylase-inhibiting antibiotic, andrimid in Serratia is regulated by Hfq and the LysR-type transcriptional regulator, AdmX.

Infections due to multidrug-resistant bacteria represent a major global health challenge. To combat this problem, new antibiotics are urgently needed and some plant-associated bacteria are a promising source. The rhizobacterium Serratia plymuthica A153 produces several bioactive secondary metabolites, including the anti-oomycete and antifungal haterumalide, oocydin A and the broad spectrum polyamine antibiotic, zeamine. In this study, we show that A153 produces a second broad spectrum antibiotic, andrimid. Using genome sequencing, comparative genomics and mutagenesis, we defined new genes involved in andrimid (adm) biosynthesis. Both the expression of the adm gene cluster and regulation of andrimid synthesis were investigated. The biosynthetic cluster is operonic and its expression is modulated by various environmental cues, including temperature and carbon source. Analysis of the genome context of the adm operon revealed a gene encoding a predicted LysR-type regulator, AdmX, apparently unique to Serratia strains. Mutagenesis and gene expression assays demonstrated that AdmX is a transcriptional activator of the adm gene cluster. At the post-transcriptional level, the expression of the adm cluster is positively regulated by the RNA chaperone, Hfq, in an RpoS-independent manner. Our results highlight the complexity of andrimid biosynthesis - an antibiotic with potential clinical and agricultural utility.