Hemolytic activity of Serratia marcescens

A cell-bound hemolytic activity was found in several strains of Serratia marcescens. One Serratia cell per ten erythrocytes was sufficient to cause complete lysis of human erythrocytes within 2 h in the liquid assay. The hemolytic activity resided in the membrane fraction and could be inactivated by incubating cells with proteases. The hemolytic activity was greatly enhanced in actively metabolizing Serratia cells and was partially controlled by the iron supply. Hemolysis was accompanied by degradation of erythrocyte membrane proteins (band 3 and 6, glycophorin) and was independent of the blood group. The exoprotease secreted by S. marcescens in large amounts was not involved in hemolysis. Comparison with various hemolytic strains of Escherichia coli showed that hemolysis of erythrocytes was more pronounced with S. marcescens than with E. coli. In contrast to hemolysis by E. coli, lysis of erythrocytes by S. marcescens was not enhanced by Ca²⁺ ions.Dedicated to Professor Dr. Gerhart Drews on the occasion of his 60th birthday     

Novel Endophytes of Rice form a Taxonomically Distinct Subgroup of Serratia marcescens

Six endophytic strains isolated from surface-sterilized rice roots and stems of different rice varieties grown in the Philippines were characterized. They were analyzed by physiological and biochemical tests, SDS-PAGE of whole-cell protein patterns, DNA-DNA hybridization and 16S rDNA sequencing. SDS-PAGE of whole-cell patterns showed that the six isolates fell into two subgroups which were similar but not identical in protein patterns to S. marcescens. The phylogenetic analysis of 16S rDNA sequences of two representative strains IRBG 500 and IRBG 501 indicated that they were closely related to S. marcescens(more than 99% identity). Physiological and biochemical tests corroborated that the isolates were highly related to each other and to S. marcescens. In cluster analysis, all six isolates were clustered together at 93% similarity level and grouped closely with Serratia marcescens at 86% similarity level. DNA-DNA hybridization studies revealed that the isolates shared high similarity levels with S. marcescens(≥86% DNA-DNA binding), indicating they belong to the same species. However, the isolates differed in several biochemical characteristics from the type strain. They produce urease and utilize urea and L(+) sorbose as a substrate, which is different from all known Serratia reference strains. These results suggest that the six endophytic isolates represent a novel, non-pigmented subgroup of S. marcescens.     

Expression of klebsiella his and nif genes in serratia marcescens,Erwinia herbicola and Proteus mirabilis

Plasmid pRD1, an R plasmid of the P incompatibility group which carries his and nif genes from Klebsiella pneumoniae in addition to drug resistance markers derived from RP4, was transferred to His^- mutants of Serratia marcescens, Erwinia herbicola and Proteus mirabilis. His⁺ transconjugants were obtained at low but different frequencies according to recipient genus. Transconjugants all acquired the drug resistance, and were Nif⁺ in S. marcescens and E. herbicola, having acetylene-reducing activities of the same order of magnitude as the parent K. pneumoniae and fixing ¹⁵N₂. No evidence for nif expression in P. mirabilis transconjugants was obtained though the nif genes were present.     

The hemolytic and cytolytic activities of Serratia marcescens phospholipase A (PhlA) depend on lysophospholipid production by PhlA

Background  

Serratia marcescens is a gram-negative bacterium and often causes nosocomial infections. There have been few studies of the virulence factors of this bacterium. The only S. marcescens hemolytic and cytotoxic factor reported, thus far, is the hemolysin ShlA.     

Serratia species isolated from plants

Using the selective caprylate-thallous agar medium, the presence ofSerratia species was systematically examined in 623 plant samples. A total of 167Serratia strains was isolated from these plant samples and identified to species and biogroups. Uniform and characteristicSerratia populations were found in figs and coconuts: (i)Serratia ficaria was recovered from most figs collected in California, Tunisia, and France; various biotypes ofS. marcescens also were found in figs; (ii) onlyS. marinorubra was recovered from coconuts bought on two continents. From plants other than figs and coconuts, representatives were isolated of all eightSerratia species we presently recognize—with a large preponderance ofS. liquefaciens andS. proteamaculans. These other plant samples fell into threeSerratia-prevalence groups: (i) vegetables-mushrooms-mosses-decaying plant material (53.8% of these samples were positive forSerratia); (ii) grasses (23.7% positive); and (iii) trees and shrubs-small plants (8.4% positive). PigmentedS. marcescens biotypes were rarely isolated from plants (except from figs). Of theS. marcescens biogroups most frequently encountered in nosocomial and iatrogenic infections of man, A3 and A4 were isolated from plants in this study, but A5/8 and TCT were not.     

Drug Resistance of Enteric Bacteria

The strains of gram-negative rod bacteria which are resistant to α-aminobenzylpenicillin and do not harbor the R factors were selected from our stock cultures of clinical origin. It was found that all strains produced β-lactamases which are species-specific in their substrate profiles and classified into three groups; 1) Typical cephalosporinase in the strains of Escherichia freundii, Aerobacter aerogenes, Arizona, Proteus morganii, Proteus rettgeri, Proteus inconstans and a strain GN633 of the Serratia group. 2) Cephalosporinase in the strains of Proteus vulgaris and a strain GN629 of the Serratia group, which has a property of penicillinase to some extent. 3) Penicillinase in the strains of Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis. It was found that cephalosporinase was generally inducible enzyme, penicillinase was constitutive, and the penicillinase synthesized by the strains carrying R factors belonged to the third group. Penicillinases of two R factors, RGN14 and RGN238 which were isolated in this laboratory and belonged to the penicillinase of the third group, were studied by comparing their substrate profiles and immunological properties. It was demonstrated that penicillinases of RGN14 and RGN238 differed each other, while the penicillinase of K. pneumoniae was quite similar to that of RGN14 both enzymologically and immunologically.     

Isolation and characterization of novel Serratia marcescens (AY927692) for pentachlorophenol degradation from pulp and paper mill waste

Seven aerobic bacterial strains were isolated from pulp paper mill waste and screened for pentachlorophenol (PCP) tolerance on PCP containing mineral salt agar medium (MSM). The organism was characterized by 16S rDNA sequencing which showed 99.7% sequence similarity with Serratia marcescens. PCP degradation was routinely monitored with spectrophotometric analysis and further confirmed by HPLC analysis. Among seven strains, ITRC S₇ was found to degrade up to 90.33% of 1.127 mM (300 mg/l) of PCP and simultaneous release of chloride ion (2.435 mM) emphasized the bacterial dechlorination in the medium in presence of glucose as an additional carbon and energy source under optimized condition within 168 h incubation. In absence of glucose bacterium was unable to utilize PCP indicating the phenomenon of co-metabolism. Bacterium was identified as S. marcescens (AY927692), was a novel and potential aerobic bacterial strain capable of degrading PCP in axenic condition. Further, this strain may be used for bioremediation of PCP containing pulp paper mill waste in the environment.     

Cell-free and cell-bound hemolytic activities of Proteus penneri determined by different Hly determinants

A collection of 45 Proteus penneri strains was characterized with respect to their hemolytic activity and representative cell-free or only cell-bound hemolysin possessing strains were chosen for further study. Extracellular Proteus penneri hemolysin, which was investigated earlier by hybridization, reacted with monospecific antiserum against alpha-hemolysin of Escherichia coli. In this paper we also show, using the colony hybridization technique, that the alpha-hemolysin-like determinant is widely distributed among Proteus penneri strains. Because one of the strains tested, which expressed a high activity of cell-bound hemolytic factor, did not carry such a Hly determinant, the presence of a second hemolysin is postulated. We cannot demonstrate any difference in hybridization patterns of alpha- and beta-hemolytic Proteus penneri strains and accumulation of the toxin molecule inside the cells was also not observed. The existence of another control mechanism, external to the hly operon, for hemolysin gene is suggested.     

Effect of Carbon Substrates on Rock Phosphate Solubilization by Bacteria from Composts and Macrofauna

Five of the 207 isolates from different composts, farm waste compost (FWC), rice straw compost (RSC), Gliricidia vermicompost (GVC), and macrofauna, showed rock phosphate (RP) solubilization in buffered medium in plate culture. When tested in RP broth medium, all five strains, Enterobacter cloacae EB 27, Serratia marcescens EB 67, Serratia sp. EB 75, Pseudomonas sp. CDB 35, and Pseudomonas sp. BWB 21, showed gluconic acid production and solubilized RP. Based on cellulose-degrading and P-solubilizing ability, two strains were selected for further studies. In the presence of different carbon sources, both strains showed a drop in pH and solubilized RP. P released was maximum with glucose (1212 and 522 μmol) and minimum with cellobiose (455 and 306 μmol) by S. marcescens EB 67 and Pseudomonas sp. CDB 35, respectively. Glucose dehydrogenase (GDH) activity was 63 and 77% with galactose and 35 and 46% with cellobiose when compared to glucose (100%) by EB 67 and CDB 35, respectively. Both strains solubilized RP in the presence of different crop residues. EB 67 and CDB 35 showed maximum cellulase activity (0.027 units) in the presence of rice straw and a mixture of rice straw and root. P solubilized from RP in the presence of pigeonpea root was 134 and 140 μmol with EB 67 and CDB 35. Significantly, these bacteria isolated from composts and macrofauna solubilized rock phosphate in the presence of various pure carbon substrates and crop residues and their importance in soil/rhizosphere conditions is discussed.     

Isolation and identification of N2-fixing microorganisms from the rhizosphere of Capparis spinosa (L.)

Four bacterial strains, Pseudomonas stutzeri var. mendocina, Comamonas sp., Agrobacterium tumefaciens biovar. 2 and Sphingobacterium sp., isolated from the rhizosphere of wild-grown caper (Capparis spinosa L.) plants were able to fix N₂ as shown by their growth in nitrogen-free medium and by the acetylene reduction test. P. stutzeri var. mendocina and Comamonas sp. contained DNA homologous to the Klebsiella pneumoniae M5a1 nifHDK genes. No hybridization was found with total DNA from either A. tumefaciens biovar. 2 or Sphingobacterium sp. using nifHDK probes from either K. pneumoniae or Rhizobium meliloti.     

Hemolysin II is more characteristic of Bacillus thuringiensis than Bacillus cereus

To investigate the distribution of the hemolysin II determinant among strains of Bacillus cereus and Bacillus thuringiensis, thirteen strains of B. cereus and fourteen strains of B. thuringiensis strains were tested for hybridization of their chromosomal DNAs with a DNA probe containing the B. cereus hemolysin II gene. In addition, the production of hemolysin II, whose activity is not inhibited by cholesterol, was tested. The presence (absence) of the hybridization response in the microorganism's genome correlated with the presence (absence) of cholesterol-unaffected hemolysin production. Only four out of thirteen B. cereus strains were found to give a positive response in hybridization experiments, whereas thirteen out of fourteen B. thuringiensis strains responded positively. DNAs from ten B. thuringiensis strains contained a 3.5 kb EcoRV fragment, which hybridized with the B. cereus hemolysin II gene probe. The 3.5 kb EcoRV DNA fragment from one of these strains (B. thuringiensis VKM-B1555) was cloned and expressed in Escherichia coli cells. The hemolysin encoded by the cloned DNA fragment was not inhibited by cholesterol and possessed all other properties of B. cereus hemolysin II. The obtained data clearly show limited distribution of hemolysin II among B. cereus strains and demonstrate that hemolysin II is more characteristic of B. thuringiensis than B. cereus.     

Characterization of chitinase‐producing Serratia and Bacillus strains isolated from insects

Chitinases (EC 3.2.1.14) are enzymes that hydrolyze chitin by cleaving β‐1,4 N‐glycosidic bonds. These enzymes have been used for multiple applications in biotechnology, especially for controlling insect pests and phytopathogenic fungi. In the present study, we isolated two chitinase‐producing bacteria strains from insects (strain SCH‐1 from Moechotypa diphysis and strain SCH‐2 from Sphedanolestes impressicollis). Serratia sp. SCH‐1 was a short, rod‐shaped facultative anaerobe, while Bacillus strain SCH‐2 was a rod‐shaped endospore‐forming anaerobe. Strains SCH‐1 and SCH‐2 were identified as Serratia sp. and Bacillus sp., respectively based on 16S rRNA gene sequencing. Strain SCH‐1 shared maximum homology (99.44%) with Serratia nematodiphila DZ0503SBS1 and Serratia marcescens subsp. sakuensis KRED. Strain SCH‐2 had a maximum homology of 99.24% with Bacillus thuringiensis ATCC 10792 and Bacillus toyonensis BCT‐7112. Serratia sp. SCH‐1 contained greater levels of saturated fatty acids, but the concentration of branched acids, especially iso‐C_15:0, was highest in Bacillus sp. SCH‐2. Serratia sp. SCH‐1 possessed chitinase activity of 1.59 unit/mg protein after 5 days of incubation in culture medium. In contrast, Bacillus sp. SCH‐2 had a maximum activity of 0.84 unit/mg protein after 4 days of incubation. Chitinase isozymes produced by Serratia sp. SCH‐1 appeared as five bands with sizes of 20, 26, 36, 45 and 54 kDa. Bacillus sp. SCH‐2 showed a chitinase isozyme profile with three bands having sizes of 36, 45 and 50 kDa on SDS‐PAGE gels.     

DNA sequence analysis of the tellurite-resistance determinant from clinical strain of Escherichia Coli and identification of essential genes

The tellurite-resistant Escherichia coli strain KL53 was found during testing of the group of clinical isolates for antibiotics and heavy metal ion resistance (Burian et al. 1990). Determinant of the tellurite resistance of the strain was located on the large conjugative plasmid pTE53 and cloned into pACYC184. Three different Te^r clones harboring pLK2, pLK18 and pLK20 were isolated (Burian et al. 1998). The smallest functional Te^r clone harboring pLK18 was chosen for further analysis. Plasmid pLK18 have been subcloned to obtain convenient DNA fragments for sequencing of tellurite-resistance determinant. Sequencing of this DNA fragments provided complete DNA sequence of the determinant, 5250 bp in size. The sequence has been compared with nucleotide and protein databank (BLAST programs) and significant homology with the three known operons coding for tellurite resistance has been found (determinat on plasmid pR478 from Serratia marcescens, on plasmid pMER610 from Alcaligenes sp. and chromosomal tellurite resistance genes from Proteus mirabilis). We identified 5 ORFs coding for 5 genes named terB to terF. The clone harboring pLK18 was subjected to the transposition with Tn1737Km to disrupt determinant of the tellurite resistance. Plasmid DNA of several clones containing pLK18 with Tn1737Km was isolated to locate the target site of Tn1737Km. Analyses showed, the genes terB, terC, terD and terE are essential for conservation of the resistance whereas the gene terF is not important in this respect.     

Assessment of antibacterial effects of flavonoids by estimation of generation times in liquid bacterial cultures

Antibacterial activities of various flavonoids, a group of natural plant substances, have been reported previously, however, there are contradictory data, published by various authors, regarding sensitivity of particular bacterial species to these compounds. These problems arose apparently because of using different methods by various researchers. Here we tested sensitivity of several bacterial species (Gram-positive: Bacillus subtilis, Micrococcus luteus, Sarcina sp. and Staphylococcus aureus; and Gram-negative: Citrobacter freundii, Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella enterica, Serratia marcescens and Vibrio harveyi) to various flavonoids: genistein and daidzein (isoflavones), apigenin (a flavone), naringenin (a flavanone) and kaempferol (a flavonol) by measurement of generation times of bacteria in liquid cultures. The presented results indicate that this simple method is adequate for unambiguous assessment of sensitivity of bacterial strains to flavonoids.     

Survey of the Distribution of Different Types of Nitrogenases and Hydrogenases in Heterocyst-Forming Cyanobactera

As a first step toward developing the methodology for screening large numbers of heterocyst-forming freshwater cyanobacteria strains for the presence of various types of nitrogenases and hydrogenases, we surveyed the distribution of these genes and their activities in 14 strains from culture collections. The nitrogenase genes include nif1 encoding a Mo-type nitrogenase expressed in heterocysts, nif2 expressed in vegetative cells and heterocysts under anaerobic conditions, and vnf encoding a V-type nitrogenase expressed in heterocysts. Two methods proved to be valuable in surveying the distribution of nitrogenase types. The first method was Southern blot hybridization of DNA digested with two different endonucleases and hybridized with nifD1, nifD2, and vnfD probes. The second method was ethane formation from acetylene to detect the presence of active V-nitrogenase. We found that all 14 strains have nifD1 genes, and eight strains also have nifD2 genes. Four of the strains have vnfD genes, in addition to nifD2 genes. It is curious that three of these four strains had similar hybridization patterns with all of the nifD1, nifD2, and vnfD probes, suggesting that there could be some bias in strains used in the present study or in strains held in culture collections. This point will need to be assessed in the future. For surveying the distribution of hydrogenases, Southern blot hybridization was an effective method. All strains surveyed had hup genes, with the majority of them also having hox genes.     

A comparative study on the phylogenetic diversity of culturable actinobacteria isolated from five marine sponge species

A cultivation-based approach was employed to compare the culturable actinobacterial diversity associated with five marine sponge species (Craniella australiensis, Halichondria rugosa, Reniochalina sp., Sponge sp., and Stelletta tenuis). The phylogenetic affiliation of the actinobacterial isolates was assessed by 16S rDNA-RFLP analysis. A total of 181 actinobacterial strains were isolated using five different culture media (denoted as M1–M5). The type of medium exhibited significant effects on the number of actinobacteria recovered, with the highest number of isolates on M3 (63 isolates) and the lowest on M1 (12 isolates). The genera isolated were also different, with the recovery of three genera on M2 and M3, and only a single genus on M1. The number of actinobacteria isolated from the five sponge species was significantly different, with a count of 83, 36, 30, 17, and 15 isolates from S. tenuis, H. rugosa, Sponge sp., Reniochalina sp., and C. australiensis, respectively. M3 was the best isolation medium for recovery of actinobacteria from S. tenuis, H. rugosa, and Sponge sp., while no specific medium preference was observed for the recovery of actinobacteria from Reniochalina sp., and C. australiensis. The RFLP fingerprinting of 16S rDNA genes digested with HhaI revealed six different patterns, in which 16 representative 16S rDNAs were fully sequenced. Phylogenetic analysis indicated that 12 strains belong to the group Streptomyces, three strains belong to Pseudonocardia, and one strain belongs to Nocardia. Two strains C14 (from C. australiensis) and N13 (from Sponge sp.) have only 96.26% and 96.27% similarity to earlier published sequences, and are therefore potential candidates for new species. The highest diversity of three actinobacteria genera was obtained from Sponge sp., though the number of isolates was low. Two genera of actinobacteria, Streptomyces, and Pseudonocardia, were isolated from both S. tenuis and C. australiensis. Only the genus of Streptomyces was isolated from H. rugosa and Reniochalina sp. Sponge species have been demonstrated here to vary as sources of culturable actinobacterial diversity, and the methods for sampling such diversity presented may be useful for improved sampling of such diversity.     

Isolation and identification of bacterial strains with insecticidal activities from Rhynchophorus ferrugineus Oliver (Coleoptera: Curculionidae)

Several pathogenic bacteria strains were isolated from dead larvae of the weevil, Rhynchophorus ferrugineus Oliver, collected on palm trees in Taiwan, China. Strains of pure cultures were temporarily named TW‐1, TW‐2, TW‐3, TW‐4, TW‐5, TW‐6, TW‐7, TW‐8, TW‐9, TW‐10, TW‐11, TW‐12, TW‐13 and TW‐14. These strains were identified by synthetically using colony morphological characters, physiological and biochemical tests and molecular biological analysis. Our results showed that these 14 isolated strains belonged to four genera and five species. The expected approximate 1400‐bp bands were obtained through 16S rDNA universal primer amplification. The sequencing results obtained from a commercial sequencing company were initially compared with a corresponding database from NCBI using BLAST, and a phylogenetic tree was constructed using mega 5.1 software. The phylogenetic results indicated that these isolated strains had more than 99% homology with type strains. To further confirm these strains, numerous physiological and biochemical indexes were confirmed. TW‐1, TW‐2, TW‐3, TW‐9, TW‐12, TW‐13 and TW‐14 were identified as Serratia marcescens; TW‐4, TW‐5, TW‐8 and TW‐10 as Staphylococcus sciuri; TW‐6 as Klebsiella pneumonia subsp. pneumonia; TW‐7 as Proteus vulgaris and TW‐11 as Proteus mirabilis. The preliminary single‐dose bioassay on fourth instar larvae showed that S. marcescens had a higher virulence at a density of 1.89 OD₆₀₀ (56.37% mortality) than the other strains. The corrected mortalities of the other strains were all less than 50%. The lowest toxicities were found for P. vulgaris and P. mirabilis where the corrected mortalities were only 28.18% and 25.57%, respectively. LC₅₀ of S. marcescens was 1.2 × 10⁷ CFU/ml inoculums. Our results indicated that S. marcescens from facultative cultivable bacterial flora isolated from R. ferrugineus could potentially be used as a microbial control agent for this widespread pest.     

Species and biotype identification ofSerratia strains associated with insects

Forty-eightSerratia strains associated with insects were, identified to species level and biotyped according to recent taxonomic schemes. Each strain was submitted to 36 biochemical tests, including 23 carbon source utilization tests. Twenty-eight strains were assigned to eight biotypes ofSerratia marcescens: A1a, A2a, and A6a (pigmented biotypes: 18 strains); and A3a, A3b, A4a, A5, and TCT (nonpigmented biotypes: 10 strains). However, biotypes A8a, A8b, and A8c, which are frequently involved in nosocomial infections, were not found in insects. Ninetten strains were identified asS. liquefaciens (S. proteamaculans) biotypes C1a (12 strains), C1c (4 strains), C1d (2 strains), and one atypicalS. liquefaciens strain. Only one strain was identified asS. marinorubra (a nonchitinolytic species). The recent emergence ofSerratia in human pathology calls for a reevaluation of the idea of usingSerratia to biologically control insects.     

Bacterial scavenging: Utilization of fatty acids localized at a solid-liquid interface

A model oligotrophic aquatic system involving localization of fatty acids on a solid surface was used to quantitate scavenging by three bacteria; Leptospira biflexa patoc 1 which adheres reversibly, pigmented Serratia marcescens EF190 which adheres irreversibly, and a non-pigmented hydrophilic mutant of EF190. The Leptospira and pigmented Serratia displayed two distinct scavenging strategies which are related to their different methods of adhesion. The Leptospira efficiently scavenged [1-¹⁴C] stearic acid from the surface in 24 h, whereas the pigmented hydrophobic Serratia initially showed a faster rate of removal but the overall rate was considerably slower than that of the Leptospira. The hydrophilic, non-pigmented Serratia required 50h incubation to remove significant amounts of the labelled fatty acid. The greater scavenging ability of the hydrophobic pigmented Serratia strain compared to the hydrophilic non-pigmented mutant could not be attributed to differences in viability of fatty acid metabolism. The hydrophobicity of the pigmented Serratia allows for firmer adhesion and greater interaction with the surface localized nutrients.     

Bacteria abundance and diversity of different life stages of Plutella xylostella (Lepidoptera: Plutellidae), revealed by bacteria culture‐dependent and PCR‐DGGE methods

Microbial abundance and diversity of different life stages (fourth instar larvae, pupae and adults) of the diamondback moth, Plutella xylostella L., collected from field and reared in laboratory, were investigated using bacteria culture‐dependent method and PCR‐DGGE analysis based on the sequence of bacteria 16S rRNA V3 region gene. A large quantity of bacteria was found in all life stages of P. xylostella. Field population had higher quantity of bacteria than laboratory population, and larval gut had higher quantity than pupae and adults. Culturable bacteria differed in different life stages of P. xylostella. Twenty‐five different bacterial strains were identified in total, among them 20 strains were presented in larval gut, only 8 strains in pupae and 14 strains in adults were detected. Firmicutes bacteria, Bacillus sp., were the most dominant species in every life stage. 15 distinct bands were obtained from DGGE electrophoresis gel. The sequences blasted in GenBank database showed these bacteria belonged to six different genera. Phylogenetic analysis showed the sequences of the bacteria belonged to the Actinobacteri, Proteobacteria and Firmicutes. Serratia sp. in Proteobacteria was the most abundant species in larval gut. In pupae, unculturable bacteria were the most dominant species, and unculturable bacteria and Serratia sp. were the most dominant species in adults. Our study suggested that a combination of molecular and traditional culturing methods can be effectively used to analyze and to determine the diversity of gut microflora. These known bacteria may play important roles in development of P. xylostella.