Biochemical Characterization and Structural Insight into Interaction and Conformation Mechanisms of Serratia marcescens Lysine Decarboxylase (SmcadA).

Inducible lysine decarboxylases (LDCs) are essential in various cellular processes of microorganisms and plants, especially under acid stress, which induces the expression of genes encoding LDCs. In this study, a novel Serratia marcesenes LDC (SmcadA) was successfully expressed in E. coli, purified and characterized. The protein had an optimal pH of 6 and a temperature of 40 °C and phylogenetic analysis to determine the evolution of SmcadA, which revealed a close relation to Enterobacteriaceae, Klebsiella sp., among others. The molecular weight of SmcadA was approximately 75 kDa after observation on SDS-PAGE and structural modeling showed the protein as a decamer, comprised of five interlinked dimers. The biocatalytic activity of the purified wild-type SmcadA (WT) was improved through site directed mutations and the results showed that the Arg595Lys mutant had the highest specific activity of 286.55 U/mg, while the Ser512Ala variant and wild-type SmcadA had 215.72 and 179.01 U/mg, respectively. Furthermore, molecular dynamics simulations revealed that interactions through hydrogen bonds between the protein residues and cofactor pyridoxal-5-phosphate (PLP) are vital for biocatalysis. Molecular Dynamics (MD) simulations also indicated that mutations conferred structural changes on protein residues and PLP hence altered the interacting residues with the cofactor, subsequently influencing substrate bioconversion. Moreover, the temperature also induced changes in orientation of cofactor PLP and amino acid residues. This work therefore demonstrates the successful expression and characterization of the purified novel lysine decarboxylase from Serratia marcesenes and provided insight into the mechanism of protein-cofactor interactions, highlighting the role of protein-ligand interactions in altering cofactor and binding site residue conformations, thus contributing to improved biocatalysis.

Genetic Analysis and Immunoelectron Microscopy of Wild and Mutant Strains of the Rubber Tree Endophytic Bacterium Serratia marcescens Strain ITBB B5-1 Reveal Key Roles of a Macrovesicle in Storage and Secretion of Prodigiosin.

Rubber tree is an economically important tropical crop. Its endophytic bacterial strain Serratia marcescens ITBB B5-1 contains an intracellular macrovesicle and red pigment. In this research, the red pigment was identified as prodigiosin by quadrupole time-of-flight mass spectrometry. Prodigiosin has a wide range of potential medical values such as anticancer and antiorgan transplant rejection. The strain ITBB B5-1 accumulated prodigiosin up to 2000 mg/L, which is higher production compared to most known Serratia strains. The formation of the macrovesicle and prodigiosin biosynthesis were highly associated and were both temporal- and temperature-dependent. A mutant strain B5-1mu that failed to produce prodigiosin was obtained by ultraviolet mutagenesis. Whole genome sequencing of wild-type and mutant strains indicated that the PigC gene encoding the last-step enzyme in the prodigiosin biosynthesis pathway was mutated in B5-1mu by a 17-bp deletion. Transmission electron microscopy analysis showed that the macrovesicle was absent in the mutant strain, indicating that formation of the macrovesicle relied on prodigiosin biosynthesis. Immunoelectron microscopy using prodigiosin-specific antiserum showed the presence of prodigiosin in the macrovesicle, the cell wall, and the extracellular vesicles, while immuno-reaction was not observed in the mutant cell. These results indicate that the macrovesicle serves as a storage organelle of prodigiosin, and secretes prodigiosin into cell envelop and culture medium as extracellular vesicles.

Simultaneous removal of hexavalent chromium and o-dichlorobenzene by isolated Serratia marcescens ZD-9.

Heavy metals-organics mixture pollution is increasingly concerned and simultaneous removal of organic pollutants and heavy metals is becoming significant. In this study, a strain was isolated from the sediment of a tannery effluent outfalls, which can remove o-dichlorobenzene (o-DCB) and Cr(VI) simultaneously. The bacterial isolate was identified as Serratia marcescens by the 16S rRNA gene sequences. The strain removed about 90% of o-DCB and more than 80% of Cr(VI) at the concentration of 1.29 g L-1 o-DCB and 20 mg L-1 Cr(VI). In the presence of concomitant pollutant o-DCB, the optimal pH (8.0) and temperature (30 °C) were determined for Cr(VI) removal. Changes of the bacterial cells and intracellular black Cr(III) sediments were observed by the TEM auxiliary analysis. The results of the FTIR spectroscopy analysis indicated that hydroxyl, amide and polysaccharides were involved in the process of Cr(VI) removal.

Wavelet-based tracking of bacteria in unreconstructed off-axis holograms.

We propose an automated wavelet-based method of tracking particles in unreconstructed off-axis holograms to provide rough estimates of the presence of motion and particle trajectories in digital holographic microscopy (DHM) time series. The wavelet transform modulus maxima segmentation method is adapted and tailored to extract Airy-like diffraction disks, which represent bacteria, from DHM time series. In this exploratory analysis, the method shows potential for estimating bacterial tracks in low-particle-density time series, based on a preliminary analysis of both living and dead Serratia marcescens, and for rapidly providing a single-bit answer to whether a sample chamber contains living or dead microbes or is empty.

Visualization of the Serratia Type VI Secretion System Reveals Unprovoked Attacks and Dynamic Assembly.

The Type VI secretion system (T6SS) is a bacterial nanomachine that fires toxic proteins into target cells. Deployment of the T6SS represents an efficient and widespread means by which bacteria attack competitors or interact with host organisms and may be triggered by contact from an attacking neighbor cell as a defensive strategy. Here, we use the opportunist pathogen Serratia marcescens and functional fluorescent fusions of key components of the T6SS to observe different subassemblies of the machinery simultaneously and on multiple timescales in vivo. We report that the localization and dynamic behavior of each of the components examined is distinct, revealing a multi-stage and dynamic assembly process for the T6SS machinery. We also show that the T6SS can assemble and fire without needing a cell contact trigger, defining an aggressive strategy that broadens target range and suggesting that activation of the T6SS is tailored to survival in specific niches.

Antimicrobial effect and membrane-active mechanism of tea polyphenols against Serratia marcescens.

In this study, we investigated the antimicrobial effect of tea polyphenols (TP) against Serratia marcescens and examined the related mechanism. Morphology changes of S. marcescens were first observed by transmission electron microscopy after treatment with TP, which indicated that the primary inhibition action of TP was to damage the bacterial cell membranes. The permeability of the outer and inner membrane of S. marcescens dramatically increased after TP treatment, which caused severe disruption of cell membrane, followed by the release of small cellular molecules. Furthermore, a proteomics approach based on two-dimensional gel electrophoresis and MALDI-TOF/TOF MS analysis was used to study the difference of membrane protein expression in the control and TP treatment S. marcescens. The results showed that the expression of some metabolism enzymes and chaperones in TP-treated S. marcescens significantly increased compared to the untreated group, which might result in the metabolic disorder of this bacteria. Taken together, our results first demonstrated that TP had a significant growth inhibition effect on S. marcescens through cell membrane damage.

Potential of chitinolytic Serratia marcescens strain JPP1 for biological control of Aspergillus parasiticus and aflatoxin.

Serratia marcescens strain JPP1 was isolated from peanut hulls in Huai'an city, Jiangsu Province, China. Its potential to inhibit the mycelial growth of Aspergillus parasiticus and the subsequent aflatoxin production was evaluated. The strain JPP1 could produce chitinase to degrade fungal cell walls, which was the main mechanism of strain JPP1 for biocontrol. Scanning electron microscopy of fungi treated with the crude chitinase revealed abnormal morphological changes. While the strain was grown in the peanut hulls-based medium, the chitinase activity reached 7.39 units. RT-PCR analysis showed that the crude chitinase repressed the transcription of genes involved in the aflatoxin gene cluster, such as aflR, aflC (pksL1), and aflO (dmtA) genes. By visual agar plate assay and tip culture method, the strain JPP1 exhibited remarkable inhibitory effect on mycelia growth (antifungal ratio >95%) and subsequent aflatoxin production (antiaflatoxigenic ratio >98%). An in vitro assay with seed coating agent of bacterial suspension showed that strain JPP1 effectively reduced fungal growth and subsequent aflatoxin production on peanut seeds, and its antagonistic effect was superior to the common agricultural fungicide of carbendazim. These characteristics suggest that S. marcescens JPP1 strain could potentially be utilized for the biological control of phytopathogenic fungi and aflatoxin in Chinese peanut main producing areas.

Differential proteome analysis of a selected bacterial strain isolated from a high background radiation area in response to radium stress.

The present study describes the response of a bacterial strain, isolated from a hot spring in an area with the highest levels of natural radiation, under radium ((226)Ra) stress. The bacterium has been characterized as a novel and efficient radium biosorbent and identified as a variant of Serratia marcescens by biochemical tests and molecular recognition. In order to gain insights into key cellular events that allow this strain to survive and undergo (226)Ra adaptation and biosorption, the strain was tested under two experimental conditions of 1000 and 6000 Bq (226)Ra stress. A proteomic approach involving two-dimensional polyacrylamide gel electrophoresis and mass spectrometry was used to identify the differentially expressed proteins under (226)Ra stress. Functional assessment of identified proteins with significantly altered expression levels revealed several mechanisms thought to be involved in (226)Ra adaptation and conferring resistant phenotype to the isolate, including general stress adaptation, anti-oxidative stress, protein and nucleic acid synthesis, energy metabolism, efflux and transport proteins. It suggests that this strain through evolution is particularly well adapted to the high background radiation environment and could represent an alternative source to remove (226)Ra from such areas as well as industrial radionuclide polluted wastewaters.

Antimicrobial activity of Chinese bayberry extract for the preservation of surimi.

BACKGROUND: Chemical preservatives such as sodium nitrite and potassium sorbate have been widely used to keep surimi products fresh. However, the potential harmfulness to human health cannot be ignored. This study was conducted to develop natural preservatives for the storage of Collichthys surimi. RESULTS: Among the eight Chinese traditional herbs and fruits, Chinese bayberry extract showed the greatest inhibitory effect against surimi spoilage bacteria Serratia marcescens and Pseudomonas aeruginosa. Moreover, N-butanol phase extract of bayberry (NB) showed the greatest activity among the different phases of bayberry extract. When Chinese bayberry extract was combined with tea polyphenol, an additive inhibitory effect was observed on growth of Hansenula anomala, Micrococcus luteus, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. Our results further indicated that the shelf life of surimi products stored at room temperature can be extended when supplemented with Chinese bayberry extract. CONCLUSION: Our results suggest that Chinese bayberry extract can be used as a natural preservative for the storage of Collichthys surimi.

Serratia marcescens strains implicated in adverse transfusion reactions form biofilms in platelet concentrates and demonstrate reduced detection by automated culture.

BACKGROUND AND OBJECTIVES: Serratia marcescens is a gram-negative bacterium that has been implicated in adverse transfusion reactions associated with contaminated platelet concentrates. The aim of this study was to investigate whether the ability of S. marcescens to form surface-attached aggregates (biofilms) could account for contaminated platelet units being missed during screening by the BacT/ALERT automated culture system. MATERIALS AND METHODS: Seven S. marcescens strains, including biofilm-positive and biofilm-negative control strains and five isolates recovered from contaminated platelet concentrates, were grown in enriched Luria-Bertani medium and in platelets. Biofilm formation was examined by staining assay, dislodging experiments and scanning electron microscopy. Clinical strains were also analysed for their ability to evade detection by the BacT/ALERT system. RESULTS: All strains exhibited similar growth in medium and platelets. While only the biofilm-positive control strain formed biofilms in medium, this strain and three clinical isolates associated with transfusion reactions formed biofilms in platelet concentrates. The other two clinical strains, which had been captured during platelet screening by BacT/ALERT, failed to form biofilms in platelets. Biofilm-forming clinical isolates were approximately three times (P<0·05) more likely to be missed by BacT/ALERT screening than biofilm-negative strains. CONCLUSION: S. marcescens strains associated with transfusion reactions form biofilms under platelet storage conditions, and initial biofilm formation correlates with missed detection of contaminated platelet concentrates by the BacT/ALERT system.

Unraveling the role of the rssC gene of Serratia marcescens by atomic force microscopy.

The product and direct role of the rssC gene of Serratia marcescens is unknown. For unraveling the role of the rssC gene, atomic force microscopy has been used to identify the surfaces of intact S. marcescens wild-type CH-1 cells and rssC mutant CH-1ΔC cells. The detailed surface topographies were directly visualized, and quantitative measurements of the physical properties of the membrane structures were provided. CH-1 and CH-1ΔC cells were observed before and after treatment with lysozyme, and their topography-related parameters, e.g., a valley-to-peak distance, mean height, surface roughness, and surface root-mean-square values, were defined and compared. The data obtained suggest that the cellular surface topography of mutant CH-1ΔC becomes rougher and more precipitous than that of wild-type CH-1 cells. Moreover, it was found that, compared with native wild-type CH-1, the cellular surface topography of lysozyme-treated CH-1 was not changed profoundly. The product of the rssC gene is thus predicted to be mainly responsible for fatty-acid biosynthesis of the S. marcescens outer membrane. This study represents the first direct observation of the structural changes in membranes of bacterial mutant cells and offers a new prospect for predicting gene expression in bacterial cells.

Prodigiosin is not a determinant factor in lysis of Leishmania (Viannia) braziliensis after interaction with Serratia marcescens D-mannose sensitive fimbriae.

In this paper, the lytic activity of two variants of Serratia marcescens against promastigotes of Leishmania braziliensis was studied. In vitro assays showed that S. marcescens variant SM365 lyses L. braziliensis promastigotes, while the variant DB11 did not. Scanning electron microscopy (SEM) revealed that S. marcescens SM365 adheres to all cellular body and flagellum of the parasite. Several filamentous structures were formed and identified as biofilms. After 120min incubation, they connect the protozoan to the developing bacterial clusters. SEM also demonstrated that bacteria, adhered onto L. braziliensis promastigote surface, formed small filamentous structures which apparently penetrates into the parasite membrane. d-mannose protects L. braziliensis against the S. marcescens SM365 lytic effect in a dose dependent manner. SM365 variant pre cultivated at 37 degrees C did not synthesize prodigiosin although the adherence and lysis of L. braziliensis were similar to the effect observed with bacteria cultivated at 28 degrees C, which produce high concentrations of prodigiosin. Thus, we suggest that prodigiosin is not involved in the lysis of promastigotes and that adherence promoted by bacterial mannose-sensitive (MS) fimbriae is a determinant factor in the lysis of L. braziliensis by S. marcescens SM365.

Visualization of flagellar interactions on bacterial carpets.

Methods for the in-depth study of the physics of microscale actuation of microfluidics environments by flagellated bacteria 'teamsters' have been developed. These methods, which include single and multi-colour fluorescent labelling and electron microscopy allow for the analysis of the effect that individual flagellar filaments have on bacterially driven microstructures, and allow for the investigation of the interaction and coordination of flagellar filaments of neighbouring bacteria on densely packed monolayers of bacteria, 'bacterial carpets'. We show that the flagella of bacteria that are immobilized on a surface often interact with each other, and that the flagella of these bacteria do not often form multi-flagella bundles that are aligned with the cell body.

Catabolite repression control of flagellum production by Serratia marcescens.

Serratia marcescens is an emerging opportunistic pathogen with a remarkably broad host range. The cAMP-regulated catabolite repression system of S. marcescens has recently been identified and demonstrated to regulate biofilm formation through the production of surface adhesions. Here we report that mutations in components of the catabolite repression system (cyaA and crp) eliminate flagellum production and swimming motility. Exogenous cAMP was able to restore flagellum production to adenylate cyclase mutants, as determined by transmission electron microscopy and PAGE analysis. A transposon-generated suppressor mutation of the crp motility defect mapped to upstream of the flhDC operon. This suppressor mutation resulted in an upregulation of flhD expression and flagellum production, indicating that flhDC expression is sufficient to restore flagellum production to crp mutants. Lastly, and contrary to a previous report, we found that flhD expression is controlled by the catabolite repression system using quantitative RT-PCR. Together, these data indicate that flagellum production is regulated by the cAMP-dependent catabolite repression system. Given the role of flagella in bacterial pathogenicity, the regulatory pathway described here may assist us in better understanding the putative role of motility in dissemination and virulence of this opportunistic pathogen.

A potential acyltransferase regulates swarming in Serratia marcescens.

Swarming in Serratia marcescens is a specialized form of bacterial populational surface migration behavior. A precocious swarming mutant S. marcescens O6 whose rssC is interrupted by mini-Tn5 transposon was obtained. The 364 amino-acid RssC showed a high identity to members of acyltransferase family. Characterization of the rssC knock-out mutant S. marcescens PC105 showed aberrant and hypervirulent phenotypes including precocious swarming behavior, reduced cell attachment ability and increase in swimming motility, flagellin synthesis, capsular polysaccharide (CPS) production, hemolysin activity, and cytotoxicity activity. Besides a more intense pattern of O-antigen sugar unit ladder, S. marcescens PC105 also showed an altered cell surface topology. A potential FlhDC binding site was identified at the rssC promoter region. In accordance, rssC transcriptional activity was up-regulated by FlhDC(Sm). Briefly, rssC is hierarchically classified as class II members in FlhDC(Sm) regulon, and reciprocally defect in rssC results in aberrant swarming and hypervirulence in S. marcescens.

The cyclic AMP-dependent catabolite repression system of Serratia marcescens mediates biofilm formation through regulation of type 1 fimbriae.

The mechanisms by which environmental carbon sources regulate biofilm formation are poorly understood. This study investigates the roles of glucose and the catabolite repression system in Serratia marcescens biofilm formation. The abilities of this opportunistic pathogen to proliferate in a wide range of environments, to cause disease, and to resist antimicrobials are linked to its ability to form biofilms. We observed that growth of S. marcescens in glucose-rich medium strongly stimulated biofilm formation, which contrasts with previous studies showing that biofilm formation is inhibited by glucose in Escherichia coli and other enteric bacteria. Glucose uptake is known to inversely mediate intracellular cyclic AMP (cAMP) synthesis through regulation of adenylate cyclase (cyaA) activity, which in turn controls fundamental processes such as motility, carbon utilization and storage, pathogenesis, and cell division in many bacteria. Here, we demonstrate that mutation of catabolite repression genes that regulate cAMP levels (crr and cyaA) or the ability to respond to cAMP (crp) confers a large increase in biofilm formation. Suppressor analysis revealed that phenotypes of a cAMP receptor protein (crp) mutant require the fimABCD operon, which is responsible for type 1 fimbria production. Consistently, fimA transcription and fimbria production were determined to be upregulated in a cyaA mutant background by using quantitative real-time reverse transcription-PCR and transmission electron microscopy analysis. The regulatory pathway by which environmental carbon sources influence cAMP concentrations to alter production of type 1 fimbrial adhesins establishes a novel mechanism by which bacteria control biofilm development.

Leishmania (Leishmania) chagasi interactions with Serratia marcescens: ultrastructural studies, lysis and carbohydrate effects.

Studies on the lysis of L. chagasi caused by the bacteria Serratia marcescens were carried out. In vitro experiments demonstrated that S. marcescens variant SM 365, a prodigiosin pigment producer, lysed this species of Leishmania but variant DB11, a nonpigmented bacteria, was unable to lyse the parasite. High concentrations of d-mannose were found to protect L. chagasi markedly diminishing the lysis by S. marcescens SM 365. Promastigotes of L. chagasi bound the lectin Concanavalin A conjugated with FITC, the fluorescence was intensely found at the base of the flagellum (flagellar pocket). Scanning electron microscopy revealed that the bacteria adherence occurred mainly in the flagellar pocket. S. marcescens SM 365 formed filamentous structures, identified as biofilms, which connect the protozoan to the developing bacterial clusters, in low concentrations of bacteria after 30 min incubation time. We suggest that bacterial mannose-sensitive (MS) fimbriae are relevant to S. marcescens SM 365 in the lysis of L. chagasi.

Trypanosoma cruzi: ultrastructural studies of adhesion, lysis and biofilm formation by Serratia marcescens.

A few days after blood meal the number of bacteria in the anterior midgut (stomach) of Rhodnius prolixus, a vector of Trypanosoma cruzi, the causative agent of Chagas' disease, increases dramatically. Many of the bloodstream trypomastigotes of the pathogenic protozoan as well as ingested erythrocytes are lysed in the stomach. Incubation of T. cruzi with Serratia marcescens variant SM365, lead to parasite lysis. In the present study, this bacterium rapidly adhered to the protozoan surface through d-mannose recognizing fimbriae and rapidly induced its complete lysis. In contrast, the DB11 variant of the same bacterial species did not adhere and did not induce protozoan lysis. Scanning and transmission electron microscopy revealed that following bacteria-protozoan attachment there is an assembly of long filamentous structures, identified as a biofilm, which connect the protozoan to the bacteria forming bacterial clusters. We conclude that parasite lysis and biofilm formation mechanisms are important for understanding parasite-microbiota interactions in the gut of insect vectors of trypanosomatids.

Regulatory roles of spnT, a novel gene located within transposon TnTIR.

The transposon TnTIR contains spnIR quorum-sensing system regulating sliding motility and the production of nuclease, biosurfactant, and prodigiosin in Serratia marcescens. Within TnTIR, a gene named spnT is upstream of and co-transcribed with spnI. SpnT is a cytoplasmic protein and its level peaks during early stationary phase. spnT over-expression resulted in inhibition of sliding motility and synthesis of prodigiosin, and biosurfactant similar to spnR. spnT but not spnR over-expression induced cell elongation and aberrant DNA replication in S. marcescens and Escherichia coli strains. In comparison with wild-type E. coli strain, over-expression of spnT in an E. coli priA and dnaC double-mutant strain did not lead to the aberrant cell morphology phenotypes, suggesting SpnT may act through the recombination-dependent DNA replication system. As spnT over-expression inhibited swarming but not swimming motility, SpnT may indirectly function as a negative regulator of surface-dependent migration and secondary metabolite production.

Colloid lithography-induced polydimethylsiloxane microstructures and their application to cell patterning.

Colloidal lithography was used to make a novel array (2-D) of micro-rings, dots, and interconnected-honeycomb structures. These geometries are controlled using the curing temperature-dependent rheological properties of the siloxane elastomer precursor. Serratia marcescens was patterned on the interconnected honeycomb microstructure demonstrating a potential application for microbioanalytical devices, microfluidics, and bio-micro-electromechanical systems.