Extracellular chitinases of mutant superproducing strain <Emphasis Type="Italic">Serratia marcescens</Emphasis> M-1

Four extracellular proteins with chitinase activity capable of binding chitin substrates have been revealed in the culture liquid of chitinase superproducing mutant strain M-1 of Serratia marcescens. Proteins were analyzed by SDS-PAGE and MALDI-TOF mass spectrometry. Based on the data obtained, the proteins were identified as typical chitinases of S. marcescens: ChiA, ChiB, ChiC, and CBP21.     

Proteolytic activity in<Emphasis Type="Italic">Serratia marcescens</Emphasis> clinical isolates

Exoproteinase production was demonstrated in 64 clinical isolates of S. marcescens. A significant relationship was found between the site of origin (autopsy material, hemocultures, various other sources), proteinase activity, and LD50 of the analyzed isolates. The number of exoproteinases varied during a 14-h incubation in batch cultures; the most frequently found was a 57.5-kDa proteinase which was observed in all analyzed strains. The exoproteinase production was shown to be related to strain virulence.     

Relationship between <Emphasis Type="Italic">Psoroptes cuniculi</Emphasis> and the Internal Bacterium <Emphasis Type="Italic">Serratia marcescens</Emphasis>

The bacterium Serratia marcescens isolated from surface-sterilised Psoroptes cuniculi was found sensitive to the antibiotic Amikacin. Mites placed in this antibiotic for 48–72 h and then washed by centrifugation were found to be alive and S. marcescens-free. Two experimental infestations were undertaken in order to verify the ability of the S. marcescens-free mites to infect and to give ear skin lesions in healthy rabbits and to evaluate the differential ability of the S. marcescens-free and S. marcescens-infected mites to give ear skin lesions. All rabbits were found to be infested, but only rabbits infested with S. marcescens-free mites presented crusts in their ears, whereas mites and/or eggs were only detected in the ear cerumen of all rabbits infested with S. marcescens-infected mites. S. marcescens was isolated only from P. cuniculi mites taken from these latter rabbits. Results indicate that P. cuniculi mites do not need S. marcescens to live and to be able to infest a healthy rabbit. In addition, S. marcescens was not isolated from eggs and newly born larvae of S. marcescens-infected P. cuniculi, demonstrating that in a population of P. cuniculi this bacterium is not transmitted transovarially.     

Synergistic biodegradation of pentachlorophenol by <Emphasis Type="Italic">Bacillus cereus</Emphasis> (DQ002384), <Emphasis Type="Italic">Serratia marcescens</Emphasis> (AY927692) and <Emphasis Type="Italic">Serratia marcescens</Emphasis> (DQ002385)

The consortium of Bacillus cereus (DQ002384), Serratia marcescens (AY927692) and Serratia marcescens (DQ002385) were used for pentachlorophenol (PCP) degradation. The consortia showed better overall removal efficiencies than single strains by utilization of PCP as a carbon and energy source confirmed by pH dependent dye indicator bromocresol purple (BCP) in mineral salt media (MSM). Mixed culture was found to degrade up to 93% of PCP (300 mg/l) as compared to single strains (62.75–90.33%), at optimized conditions (30 ± 1°C, pH 7 ± 0.2, 120 rpm) at 168 h incubation. PCP degradation was also recorded at 20°C (62.75%) and 37°C (83.33%); pH 6 (70%) and pH 9 (75.16%); 50 rpm (73.33%) and 200 rpm (91.63%). The simultaneous release of chloride ion up to 90.8 mg/l emphasized the bacterial dechlorination in the medium. GC–MS analysis revealed the formation of low molecular weight compound, i.e., 6-chlorohydroxyquinol, 2,3,4,6-tetrachlorophenol and tetrachlorohydroquinone, from degraded sample as compared to control.     

Diversity and specificity in the interaction between <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> and the pathogen <Emphasis Type="Italic">Serratia marcescens</Emphasis>

Background  

Co-evolutionary arms races between parasites and hosts are considered to be of immense importance in the evolution of living organisms, potentially leading to highly dynamic life-history changes. The outcome of such arms races is in many cases thought to be determined by frequency dependent selection, which relies on genetic variation in host susceptibility and parasite virulence, and also genotype-specific interactions between host and parasite. Empirical evidence for these two prerequisites is scarce, however, especially for invertebrate hosts. We addressed this topic by analysing the interaction between natural isolates of the soil nematode Caenorhabditis elegans and the pathogenic soil bacterium Serratia marcescens.     

Isolation of <Emphasis Type="Italic">Serratia marcescens</Emphasis> involved in chitin degradation in the bulb mite <Emphasis Type="Italic">Rhizoglyphus robini</Emphasis>

Cymbomonas tetramitiformis is a peculiar green alga that unites in one cell the abilities of photosynthesis and phagocytosis, which makes it a very useful model for the study of the evolution of plastid endosymbiosis. We have pondered over this issue and propose an evolutionary scenario of trophic strategies in eukaryotes, including primary and secondary plastid endosymbioses. C. tetramitiformis is a prototroph, just like the common ancestor of Archaeplastida was, and can synthesize most small organic molecules contrary to other eukaryotic phagotrophs, e.g. some metazoans, amoebozoans, and ciliates, which have not evolved tight endosymbiotic relationships. In order to establish a permanent photosynthetic endosymbiont they do not have to become prototrophs, but have to acquire the genes necessary for plastid retention via horizontal (including endosymbiotic) gene transfer. Such processes occurred successfully in the ancestors of eukaryotes with permanent secondary plastids and thus led to their great diversification. The preservation of phagocytosis in Cymbomonas (and some other prasinophytes as well) seems to result from nutrient deficiency in their oligotrophic habitats. This forces them to supplement their diet with phagocytized prey, in contrasts to the thecate amoeba Paulinella chromatophora, which also successfully transformed cyanobacteria into permanent organelles. Although Paulinella endosymbionts were acquired very recently in comparison to primary plastids, Paulinella has lost the ability to phagocytose, most probably due to the fact that it inhabits nutrient-rich environments, which renders the phagotrophy nonessential.     

Chitinases from <Emphasis Type="BoldItalic">Serratia marcescens</Emphasis> Nima

Chitinolytic activity of Serratia marcescens Nima (130 U ml⁻¹) was up to 43 times higher than those produced by other S. marcescens strains. This strain synthesized an endochitinase (Chi-60), an exochitinase (Chi-50) and a novel N-acetylglucosaminidase. This latter showed two putative isoforms (Chi-180.5 and Chi-180.8) with isoelectric points of 5 and 8.1, respectively.     

Simultaneous removal of hexavalent chromium and <Emphasis Type="Italic">o</Emphasis>-dichlorobenzene by isolated <Emphasis Type="Italic">Serratia marcescens</Emphasis> 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.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Optimal conditions for chitinase production by<Emphasis Type="Italic">Serratia marcescens</Emphasis>

A chitinase-producing bacterium was isolated from seashore mud around Beobseongpo in Chunmam province through the use of a selective enrichment culture. The best chitinase producing strain was isolated and identified asSerratia marcescens KY from its characteristics. For effective production of chitinase, optimum pH, temperature, and agitation speed were investigated in flask cultures. The optimum pH usingSerratia marcescens KY was between pH 6 and 7 and the chitinase produced was 37.9 unit/mL. On the other hand, the optimal pH of theSerratia marcescens ATCC 27117 was 7.5, and the produced amount of chitinase was 35.2 unit/mL. The optimal temperature for chitinase production forSerratia marcescens KY andSerratia marcescens ATCC 27117 was 30°C. The cell growth pattern at different temperature was almost identical to the chitinase production. To investigate the optimal shaking speed under optimal culture, speeds were varied in the range of 0≈300 rpm. The maximum production of chitinase was carried at 200 rpm although the cell growth was the highest at 150 rpm. It indicates that oxygen adjustment is required for the maximum chitinase production. Using optimal conditions, batch cultures for comparingSerratia marcescens KY andSerratia marcescens ATCC 27117 were carried out in a 5 L fermentor. The oxygen consumption was increased with the increase of culture. Especially, at 120 h of cultureSerratia marcescens KY andSerratia marcescens ATCC 27117 produced 38.3 unit/mL, and 33.5 unit/mL, respectively.     

Nuclease Biosynthesis and Growth of <Emphasis Type="Italic">Serratia marcescens</Emphasis> in the Presence of 2-(<Emphasis Type="Italic">p</Emphasis>-Aminobenzenesulfonamide)-thiazole

The biosynthesis of nuclease in Serratia marcescens has been studied under the conditions of purine synthesis inhibition with 2-(p-aminobenzenesulfonamide)-thiazole. The addition of this sulfonamide to S. marcescens at different growth stages is found to inhibit both culture growth and nuclease synthesis.__________Translated from Mikrobiologiya, Vol. 74, No. 3, 2005, pp. 365–369.Original Russian Text Copyright © 2005 by Starshinova, Filimonova.     

Production,formulation and antagonistic activity of the biocontrol like-yeast <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> against <Emphasis Type="Italic">Penicillium expansum</Emphasis>

Aureobasidium pullulans (de Bary) Arnaud (Ach 1-1) was grown in a glucose fed-batch fermentor to 106 g dry wt l⁻¹ in 48 h. The cells were dried in a fluidized bed dryer with a final viability of 62%. After 7 months at 4°C, the viability was 28% of the initial value (= 2.3 × 10¹⁰ c.f.u. g⁻¹ dry matter). A protection level of 89% was achieved with the biomass preparation at 1 × 10⁸ c.f.u. ml⁻¹ after 28 and 7 days for apples stored respectively at 5 and 25°C against Penicillium expansum. Our process is suitable to produce large quantities of the strain Ach 1-1 as biological control agent for apple preservation.     

A novel medium for the enhanced cell growth and production of prodigiosin from <Emphasis Type="Italic">Serratia marcescens</Emphasis> isolated from soil

Background

Prodigiosin produced by Serratia marcescens is a promising drug owing to its reported characteristics of having antifungal, immunosuppressive and antiproliferative activity. From an industrial point of view the necessity to obtain a suitable medium to simultaneously enhance the growth of Serratia marcescens and the pigment production was the aim of this work. The usage of individual fatty acid as substrate in industries would be cost-effective in the long run and this paved the way for us to try the effect of different fatty acid-containing seeds and oils of peanut, sesame and coconut as source of substrate.

Results

The addition of sugars only showed slight enhancement of prodigiosin production in nutrient broth but not in fatty acid containing seed medium. The powdered peanut broth had supported better growth of Serratia marcescens and higher yield of prodigiosin when compared with the existing nutrient broth and peptone glycerol broth. A block in prodigiosin production was seen above 30°C in nutrient broth, but the fatty acid seed medium used by us supported prodigiosin production upto 42°C though the yields were lower than what was obtained at 28°C. From the results, the fatty acid form of carbon source has a role to play in enhanced cell growth and prodigiosin production.

Conclusion

We conclude by reporting that the powdered and sieved peanut seed of different quality grades were consistent in yielding a fourty fold increase in prodigiosin production over the existing media. A literature survey on the composition of the different media components in nutrient broth, peptone glycerol broth and the fatty acid containing seeds and oils enabled us to propose that the saturated form of fatty acid has a role to play in enhanced cell growth and prodigiosin production. This work has also enabled us to report that the temperature related block of prodigiosin biosynthesis varies with different media and the powdered peanut broth supports prodigiosin production at higher temperatures. The medium suggested in this work is best suitable from an industrial point of view in being economically feasible, in terms of the higher prodigiosin yield and the extraction of prodigiosin described in this paper is simple with minimal wastage.

     

Role of <Emphasis Type="Italic">Serratia marcescens</Emphasis> ACE2 on diesel degradation and its influence on corrosion

A facultative anaerobic species Serratia marcescens ACE2 isolated from the corrosion products of diesel transporting pipeline in North West, India was identified by 16S rDNA sequence analysis. The role of Serratia marcesens ACE2 on biodegradation of diesel and its influence on the corrosion of API 5LX steel has been elucidated. The degrading strain ACE2 is involved in the process of corrosion of steel API 5LX and also utilizes the diesel as an organic source. The quantitative biodegradation efficiency (BE) of diesel was 58%, calculated by gas-chromatography–mass spectrum analysis. On the basis of gas-chromatography–mass spectrum (GC–MS), Fourier Transform infrared spectroscopy (FTIR) and X-ray diffractometer (XRD), the involvement of Serratia marcescens on degradation and corrosion has been investigated. This basic study will be useful for the development of new approaches for detection, monitoring and control of microbial corrosion.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Role of Arabidopsis <Emphasis Type="Italic">ABF1</Emphasis>/<Emphasis Type="Italic">3</Emphasis>/<Emphasis Type="Italic">4</Emphasis> during <Emphasis Type="Italic">det1</Emphasis> germination in salt and osmotic stress conditions

Key message

Arabidopsis det1 mutants exhibit salt and osmotic stress resistant germination. This phenotype requires HY5, ABF1, ABF3, and ABF4.

Abstract

While DE-ETIOLATED 1 (DET1) is well known as a negative regulator of light development, here we describe how det1 mutants also exhibit altered responses to salt and osmotic stress, specifically salt and mannitol resistant germination. LONG HYPOCOTYL 5 (HY5) positively regulates both light and abscisic acid (ABA) signalling. We found that hy5 suppressed the det1 salt and mannitol resistant germination phenotype, thus, det1 stress resistant germination requires HY5. We then queried publically available microarray datasets to identify genes downstream of HY5 that were differentially expressed in det1 mutants. Our analysis revealed that ABA regulated genes, including ABA RESPONSIVE ELEMENT BINDING FACTOR 3 (ABF3), are downregulated in det1 seedlings. We found that ABF3 is induced by salt in wildtype seeds, while homologues ABF4 and ABF1 are repressed, and all three genes are underexpressed in det1 seeds. We then investigated the role of ABF3, ABF4, and ABF1 in det1 phenotypes. Double mutant analysis showed that abf3, abf4, and abf1 all suppress the det1 salt/osmotic stress resistant germination phenotype. In addition, abf1 suppressed det1 rapid water loss and open stomata phenotypes. Thus interactions between ABF genes contribute to det1 salt/osmotic stress response phenotypes.

     

Purification and characterization of chitinases from <Emphasis Type="Italic">Paecilomyces</Emphasis><Emphasis Type="Italic">variotii</Emphasis> DG-3 parasitizing on <Emphasis Type="Italic">Meloidogyne incognita</Emphasis> eggs

Two extracellular chitinases were purified from Paecilomyces variotii DG-3, a chitinase producer and a nematode egg-parasitic fungus, to homogeneity by DEAE Sephadex A-50 and Sephadex G-100 chromatography. The purified enzymes were a monomer with an apparent molecular mass of 32 kDa (Chi32) and 46 kDa (Chi46), respectively, and showed chitinase activity bands with 0.01% glycol chitin as a substrate after SDS-PAGE. The first 20 and 15 N-terminal amino acid sequences of Chi32 and Chi46 were determined to be Asp-Pro-Typ-Gln-Thr-Asn-Val-Val-Tyr-Thr-Gly-Gln-Asp-Phe-Val-Ser-Pro-Asp-Leu-Phe and Asp-Ala-X-X-Tyr-Arg-Ser-Val-Ala-Tyr-Phe-Val-Asn-Trp-Ala, respectively. Optimal temperature and pH of the Chi32 and Chi46 were found to be both 60°C, and 2.5 and 3.0, respectively. Chi32 was almost inhibited by metal ions Ag⁺ and Hg²⁺ while Chi46 by Hg²⁺ and Pb²⁺ at a 10 mM concentration but both enzymes were enhanced by 1 mM concentration of Co²⁺. On analyzing the hydrolyzates of chitin oligomers [(GlcNAc) _n , n = 2–6)], it was considered that Chi32 degraded chitin oligomers as an exo-type chitinase while Chi46 as an endo-type chitinase.     

Activity dynamics of potential marker enzymes of <Emphasis Type="Italic">Serratia marcescens</Emphasis> cytoplasm and periplasm

Activity dynamics of glucose-6-phosphate dehydrogenase, alkaline phosphatase, β-galactosidase and β-lactamase in the course of growth and development of Gram-negative bacteria Serratia marcescens was studied. Glucose-6-phosphate dehydrogenase can serve as a marker of cytoplasm and be also used as a marker of plasma membrane continuity. Alkaline phosphatase is a marker of periplasm. Glucose-6-phosphate dehydrogenase, β-lactamase and β-galactosidase can be additionally used as markers of the outer membrane continuity of microbial cells.