Hemolysin as a marker for Serratia

All Serratia marcescens strains (total of 33) of different sources were hemolytic including clinical strains previously classified as being nonhemolytic. DNA fragments of the two hemolysin genes hybridized with the chromosomal DNA of S. marcescens, S. liquefaciens, S. kiliensis, S. grimesii, S. proteamaculans, S. plymutica, S. rubridaea which were also hemolytic. The restriction pattern of the hemolysin locus differed in each strain. S. ficaria and S. marinorubra expressed a different hemolysin which was much smaller than the S. marcescens hemolysin since it diffused through dialysis membranes. The DNA of the latter strains did not hybridize with the S. marcescens hemolysin DNA probes. Some S. marcescens strains, S. kiliensis and S. liquefaciens also expressed in addition the small hemolysin. No hybridization was found with DNA of Escherichia coli, Salmonella typhimurium, Proteus mirabilis, Proteus vulgaris, Citrobacter freundii, Enterobacter cloacae, Klebsiella arerogenes, Klebsiella pneumoniae, Shigella dysenteriae, Yersinia enterocolitica, Yersinia pseudotuberculosus, Listeria sp., Aeromonas sp., Legionella sp. and a Meningococcus sp., indicating that the hemolysin DNA probes are specific for Serratia, or that the hemolysin genes occur rarely in genera other than Serratia.     

Action of hexaamminecobalt on the activity of Serratia marcescens nuclease

Using CD spectroscopic and kinetic analysis, a refined mechanism of Co(NH₃) ₆ ³⁺ action on activity of Serratia marcescens nuclease was elucidated. The mechanism was identical with previously found mechanisms of Mg²⁺ and C₇H₅O₂Hg⁺. Similarly to Mg²⁺ and C₇H₅O₂Hg⁺, Co(NH₃) ₆ ³⁺ binding to the DNA substrate induced changes in the secondary structure which resulted in changes of the enzymatic activity of the S. marcescens nuclease. Upon binding of 0.03 Co(NH₃) ₆ ³⁺ per DNA phosphate, highly polymerized DNA displayed A-form characteristics. The DNA transition from B-form to A-form intermediate was followed by a decrease of the nuclease activity. The diminishing nuclease activity was consistent with diminishing values of Km and Kcat. Co(NH₃)₆ ³⁺ binding to the highly polymerized DNA caused a 1.7–2.8-fold decrease in Km, and 13.3–19.9 decrease in Vmax compared with Mg-DNA complex. A vast excess of Co(NH₃)₆ ³⁺ did not affect the activity of S. marcescens nuclease if the DNA in the assay mixture remained in its B-form conformation. Preincubation of S. marcescens nuclease with Co(NH₃)₆ ³⁺ did not influence the tertiary structure of the enzyme.     

Lipopolysaccharide is the receptor for kappa phage inSerratia marcescens

Kappa phage active onSerratia marcescens can form plaques on white and red strains with identical efficiencies. To identify the kappa phage receptor, the inactivation of the phage was studied after incubation with several bacterial subcellular fractions. The experiments demonstrated that kappa phage adsorbs to outer membrane fractions of susceptible cells. Proteinase K did not affect the rate of inactivation. Lipopolysaccharide proved to be the primary receptor for kappa phage. Prodigiosin content of the lipopolysaccharide fraction was low.     

Molecular characterization of polyphosphate kinase (ppk) gene from Serratia marcescens

To understand the mechanism of phosphate accumulation, a gene encoding polyphosphate kinase (PPK) was cloned from the genomic library of Serratia marcescens by Southern hybridization. From the nucleotide sequence of a 4 kb DNA fragment, an open reading frame of 2063 nucleotides was identified encoding a protein of 686 amino acids with molecular mass of 70 kDa. The potential CRP binding site and pho box sequence were found upstream of the putative promoter in the regulatory region. The expression of PPK resulted in the formation of inclusion bodies and the product was active at low temperature. The E. coli strain harboring plasmid pSPK5 with ppk gene increased enzyme activity of polyphosphate kinase, resulting in increased accumulation of polyphosphate in E. coli.     

Polyhydroxybutyrate accumulation by a Serratia sp.

A strain of Serratia sp. showed intracellular electron-transparent inclusion bodies when incubated in the presence of citrate and glycerol 2-phosphate without nitrogen source following pre-growth under carbon-limitation in continuous culture. About 1.3 mmol citrate were consumed per 450 mg biomass, giving a calculated yield of maximally 55% of stored material per g of biomass dry wt. The inclusion bodies were stained with Sudan Black and Nile Red (NR), suggesting a lipid material, which was confirmed as polyhydroxybutyrate (PHB) by analysis of molecular fragments by GC and by FTIR spectroscopy of isolated bio-PHB in comparison with reference material. Multi-parameter flow cytometry in conjunction with NR fluorescence, and electron microscopy, showed that not all cells contained heavy PHB bodies, suggesting the potential for increasing the overall yield. The economic attractiveness is enhanced by the co-production of nanoscale hydroxyapatite (HA), a possible high-value precursor for bone replacement materials.     

Characterization of imipenem-resistant Serratia marcescens producing IMP-type and TEM-type beta-lactamases encoded on a single plasmid

To evaluate the roles of blaIMP and blaTEM genes in the resistance of Serratia marcescens against beta-lactams and to find the spreading ways of these genes, 19 clinical isolates of imipenem-resistant Serratia marcescens were analyzed. Six strains bore blaIMP and blaTEM genes on a single plasmid, as confirmed by transferring resistance determinants via conjugation and transformation, and by detecting bla genes with PCR analysis. The six strains showed two different genomic patterns on pulsed-field gel electrophoresis. All the transconjugants and transformants gained high-level resistance to ampicillin, cephalexin, cefoxitin and cefotaxime, and showed a reduced susceptibility to imipenem, but maintained full susceptibility to aztreonam. In addition, the expressions of blaIMP and blaTEM genes were constitutive, either in Serratia marcescens clinical isolates or in their transconjugants and transformants. These findings may explain the rapid spread of the above resistance determinants among Enterobacteriaceae via transmissible plasmids in the clinical setting.     

Molecular cloning and purification of an endochitinase from Serratia marcescens (Nima)

An endochitinase gene from the Serratia marcescens Nima strain (chiA Nima) was cloned, sequenced, and expressed in Escherichia coli DH5αF′, and the recombinant protein (ChiA Nima) was purified by hydrophobic interaction chromatography. chiA Nima contains an open reading frame (ORF) that encodes an endochitinase with a deduced molecular weight and an isoelectric point of 61 kDa and 6.84, respectively. A sequence at the 5′-end was identified as a signal peptide, recognized by Gram-negative bacteria transport mechanism. Comparison of ChiA Nima with other chitinases revealed a modular structure formed by the catalytic domain and a putative chitin-binding domain. The purified chitinase was able to hydrolyze both trimeric and tetrameric fluorogenic substrates, but not a chitobiose analog substrate. ChiA Nima showed high enzymatic activity within a broad pH range (pH 4.0–10.0), with a peak activity at pH 5.5. The optimal temperature for enzymatic activity was detected at 55°C.     

Serratia marcescens as a rapid indicator of Microctonus hyperodae oviposition activity in Listronotus maculicollis and potential application of the technique to host-specificity testing

Listronotus maculicollis (Dietz) (Coleoptera: Curculionidae) is a potential novel host of the braconid parasitoid Microctonus hyperodae Loan, but initial studies have shown that levels of parasitism are lower than in the natural host L. bonariensis (Kuschel). A novel bacterial indicator test was used to determine whether the lower level of parasitism was due to behavioural factors, lack of oviposition, or host resistance. The incidence of ovipositor penetration by the parasitoid M. hyperodae into adult L. maculicollis was measured by immersing the ovipositor of the parasitoid in the facultative pathogen, Serratia marcescens Bizio. Adult weevils were then exposed to parasitoids for up to 72 h and rapid mortality used as an indicator of oviposition penetration. Survival was assessed after six days and surviving weevils were dissected and examined for parasitoid larvae. Mortality among L. maculicolis exposed to parasitoids treated with S. marcescens was significantly higher (P<0.001) than the controls but significantly lower (P<0.001) than in the natural host, L. bonariensis. Dissection of weevils exposed to uncontaminated parasitoids revealed that parasitism in L. maculicolis was significantly (P<0.001) less than parasitism in L. bonariensis. Serratia marcescens-induced mortality plus parasitism of surviving weevils in the parasitoid plus bacteria treatments produced a similar overall effect. Application of bacteria to the parasitoid ovipositor provided a rapid, simple test for ovipositor penetration, which shows potential for separation of behavioural and physiological defence mechanisms in parasitoid/host range studies.     

The structure of the polysaccharide part of the LPS from Serratia marcescens serotype O19, including linkage region to the core and the residue at the non-reducing end

The structure of the LPS from Serratia marcescens serotype O19 was investigated. Deamination of the LPS released the O-chain polysaccharide together with a fragment of the core oligosaccharide. The following structure of the product was determined by NMR spectroscopy, mass spectrometry, and chemical methods: [carbohydrate structure: see text] The main polymer consists of a repeating disaccharide V-U and is present on average of 18 units per chain as estimated by integration of signals in the NMR spectra. The residue O corresponds to the primer, which initiates biosynthesis of the O-chain, and an oligomer of a disaccharide R-S is an insert between the primer and the main polymer. The polysaccharide has a beta-Kdo residue at the non-reducing end, a feature similar to that observed previously in the LPS from Klebsiella O12.     

Hydrolysis of Phosphoester Bond by the Heme-Independent Chloroperoxidase from Serratia marcescens

Heme- and metal-independent chloroperoxidase from Serratia marcescens W 250 is shown to be capable of catalyzing the p-nitrophenyl phosphate hydrolysis. The parameters of the phosphatase reaction are determined and inhibitors and activators of the process are found. A hypothetical mechanism of the hydrolysis of phosphoesters by heme- and metal-independent haloperoxidases is suggested.     

Decolourization of synthetic dyes by a newly isolated strain of Serratia marcescens

A novel dye-decolourizing strain of the bacterium Serratia marcescens efficiently decolourized two chemically different dyes Ranocid Fast Blue (RFB) and Procion Brilliant Blue-H-GR (PBB-HGR) belonging respectively to the azo and anthraquinone groups. Extracellular laccase and manganese peroxidase (MnP) activity were detected during dye decolourization. The involvement of MnP activity was found in the decolourization of both dyes. More than 90% decolourization of PBB-HGR and RFB was obtained on days 8 and 5, respectively at 26 °C under static conditions at pH 7.0. MnP activity was increased by the addition of Mn²⁺ · At 50 M Mn²⁺, high MnP (55.3 U/ml) but low laccase activity (8.3 U/ml) was observed. Influence of oxalic acid on MnP activity was also observed.     

High production of prodigiosin by Serratia marcescens grown on ethanol

Serratia marcescens S389, isolated as an ethanol-utilizing bacterium, produced prodigiosin at up to 3 mg ml^–1 when grown on ethanol and with the omission of inorganic phosphate and NaCl from the medium. This yield was some 200-fold greater than that previously reported.     

Pleiotropic consequences of mutations towards antibiotic-hypersensitivity in Serratia marcescens

Various mutants (oxa ^s ) were isolated from Serratia marcescens SM-6 by selecting for hypersensitivity towards oxacillin. All mutants found are highly pleiotropic and able to yield spontaneous revertants which behave like the wild-type. Mutant W 1421 mostly studied shows the following phenotypic properties not found in the wild-type: (1) The growth is hypersensitive to various antibiotics, detergents and dyes which differ remarkably in their chemical structure and antibacterial action-mechanism, (2) the cells can be easily solubilized by 0.05% Sodium-dodecylsulfate, (3) the cells allow the adsorption of the roughmutant specific Salmonella phage 6SR, (4) strong cellular binding of crystal violet, (5) agglutination of the cells in 0.3% auramin solution and (6) reduced formation of red pigment. Strain W 1421 is assumed to be a lipopolysaccharide-defective mutant. The outer membrane of mutant W 1421 analyzed by Sodiumdodecylsulfate-polyacrylamide gel electrophoresis possesses a single protein less than that of the wild-type. Mutant W 1421 is further characterized by its low exolipase activity; exoprotease and exonuclease activities are as in the wild-type. This specific exoenzyme deficiency can be overcome either by backmutation to oxacillin-resistance or by growing mutant W 1421 in a medium supplemented with certain non-metabolizable polysaccharides, e.g. glycogen or pectin B. Both polysaccharides increase the exolipase activity of the wild-type too.List of Abbreviations amp ampicillin - LPS lipopolysaccharide - MIC minimal inhibitory concentration - NB nutrient broth - oxa oxacillin - str streptomycin - TBY tryptone broth with yeast extract - SDS sodium-dodecylsulfate - OD optical density This paper is dedicated to Prof. Dr. R. W. Kaplan, University of Frankfurt/M., on the occasion of his 65th birthday     

Inducibility of biotransformation enzymes in Serratia marcescens

A significant increase in the levels of electron transport components and biotransformation enzymes was observed when cells of Serratia marcescens were incubated for 3 h or cells grown for 8 h (exponential growth phase) in synthetic medium containing either 1.0% ethanol or 5.0% veratrole when compared to peptone-glucose medium. Phenobarbital was found to be a poor inducer of monooxygenase.     

Pyrimidine biosynthesis in Serratia marcescens: Polypeptide interactions of three nonsequential enzymes

Orotidine-5-monophosphate pyrophosphorylase (OMPppase, E.C. 2.4.2.10) and orotidylate decarboxylase (OMPdecase, E.C. 4.1.1.23) were purified from Serratia marcescens HY. These enzymes required physical association for maximal catalytic activities and formed a fragile complex with dihydroorotase (DHOase, E.C. 3.5.2.3.). OMPppase reversibly lost 50% of its activity upon separation from DHOase. The kinetic characteristics of OMPppase were modified by this separation. In the presence of DHOase, the K _ms for PRPP and orotate were stoichiometric: 2.3×10^–6 m and 2.6×10^–6 m, respectively. Following separation, the K _ms were significantly different: 1.3 × 10^–6 m for PRPP and 4.1×10^–6 m for orotate. OMPppase and OMPdecase could be reversibly separated by acrylamide gel electrophoresis, but the separation was accompanied by a loss of catalytic efficiency for both enzymes. DHOase readily associated into multiple molecular forms and could not be purified. The DHOase-OMPppase-OMPdecase interactions demonstrate that a weakly aggregated, multifunctional enzyme complex participates in the biosynthesis of pyrimidine nucleotides in S. marcescens. This unique association of nonsequential biosynthetic enzymes may represent a larger complex which provides a channeling or regulatory unit.This work was supported by grants from the National Science Foundation (NSF GB 5811) and the Office of Naval Research (Nonr 4413). One of us (J.W.) was a National Science Foundation Graduate Fellow.     

Flavonoids and Growth Hormones Influence Endophytic Colonization and in Planta Nitrogen Fixation by a Diazotrophic Serratia sp. in Rice

Summary Several endophytic diazotrophs were isolated from cultivars of rice and screened for their diazotrophy by nitrogenase assay and amplification of partial nifH gene. Ability of one of the diazotrophic endophytes, Serratia sp. (isolate EDA2 from cultivar ADT36) to colonize the rice seedlings grown in the presence of flavonoids and growth hormones, under gnotobiotic condition was assessed in cultivar ADT36 using a strain marked with transposon-based egfp and Km ^r . The endophytic colonization was monitored through re-isolation from different parts of rice seedlings in LB+Km plates. Addition of the flavonoids quercetin and diadzein to the growth medium increased the extent of endophytic colonization of the conjugant in rice seedlings by colonizing throughout the plant. Population and in planta nitrogenase activity of Serratia in rice seedlings were significantly increased by addition of flavonoids, quercetin and diadzein, whereas growth hormones, IAA and NAA reduced the efficiency of Serratia. The inoculation of Serratia sp. with flavonoids increased the plant biomass and biochemical constituents of rice seedlings under controlled condition.     

Study of the Mechanism of Action of p-Chloromercuribenzoate on Endonuclease from the Bacterium Serratia marcescens

The mechanism of action of p-chloromercuribenzoate (PCMB) on Serratia marcescens nuclease was investigated. The analysis showed that PCMB forms complexes with DNA. Binding of C₇H₅O₂Hg⁺ to DNA changes the secondary structure of the DNA. These changes alter the enzymatic activity of S. marcescens nuclease, which was previously found to be sensitive to the secondary structure of the substrates. The nuclease activity was either suppressed or stimulated in the presence of PCMB depending on the C₇H₅O₂Hg⁺ to nucleotide equivalent ratio. Binding of C₇H₅O₂Hg⁺ to DNA did not form an abortive enzyme–substrate complex. Binding of Mg²⁺ to the C₇H₅O₂Hg–DNA complex caused appropriate changes in secondary structure of the substrate. Since Mg²⁺ and C₇H₅O₂Hg⁺, though differing in the type of metal cation, are similar in their mechanisms of influence on enzymatic activity of S. marcescens nuclease, the identity of other metal-containing effectors in their mechanism of action on Serratia marcescens nuclease is assumed.     

Extracellular haemolytic activity of Serratia marcescens

Blood agar medium with dialysis membrane mounted between two layers of agar was applied to study the haemolytic activity of 28 strains of Serratia marcescens. Two kinds of lytic substances differing with their ability to pass through dialysis membrane were found. Haemolytic activity was not detected in cell-free filtrates from liquid cultures. The discrepancies between haemolytic activity in blood agar media and activity of liquid cultures were observed. Stable attachment of bacterial cells to the erythrocytes was not necessary to lysis. The possibility of extracellular haemolysin is discussed.     

Strategy for the Improvement of Prodigiosin Production by a Serratia marcescens Mutant through Fed-Batch Fermentation

A Serratia marcescens mutant for prodigiosin production was obtained by u.v. mutation with rational screening methods and a two-step feeding strategy was used to increase its productivity. In flasks, the mutant strain B6 gave a 2.8-fold higher prodigiosin production than that of the parent strain with glycerol as a carbon source. In a 5-l bioreactor, with a two-step feeding strategy in which glucose was selected as the initial carbon source in the fermentation media and glycerol was fed as a ‘prodigiosin inducer’, it gave a 7.8 times higher prodigiosin production (583 mg/l) than the parent stain with the original cultivation mode.