Identification of motile Aeromonas strains with the MicroScan WalkAway system in conjunction with the combo negative type 1S panels

This study was performed to compare the MicroScan WalkAway automated identification system in conjunction with the new MicroScan Combo Negative type 1S panels with conventional biochemical methods for identifying 85 environmental, clinical, and reference strains of eight Aeromonas species.     

Diversity of antifungal and plant-associated Serratia plymuthica strains

A total of 21 plant-associated Serratia plymuthica strains were characterized phenotypically by their nutritional patterns, susceptibility to antibiotics, antifungal and haemolytic properties, and genotypically by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA, PCR fingerprints using BOX primers (BOX-PCR) and pulsed-field gel electrophoresis (PFGE) after digestion with SpeI. All of the investigated strains demonstrated antifungal activity in vitro against fungal pathogens while only six strains produced the antifungal antibiotic prodigiosin. Haemolytic activity and antibiotic resistance patterns were investigated to assess the risk associated with the use of isolates in plant protection. The strains were haemolytic at human-relevant temperatures. The level of resistance to antibiotics was low. This work shows that BOX-PCR and PFGE are useful fingerprinting methods to characterize Ser. plymuthica strains, although the discriminatory effect between the two methods differed. Computer-assisted analysis of phenotypic and genotypic features demonstrated relationships between the origin of isolation, the production of prodigiosin and the molecular fingerprint.     

Enhancing the Thermostability of Serratia plymuthica Sucrose Isomerase Using B-Factor-Directed Mutagenesis

The sucrose isomerase of Serratia plymuthica AS9 (AS9 PalI) was expressed in Escherichia coli BL21(DE3) and characterized. The half-life of AS9 PalI was 20 min at 45°C, indicating that it was unstable. In order to improve its thermostability, six amino acid residues with higher B-factors were selected as targets for site-directed mutagenesis, and six mutants (E175N, K576D, K174D, G176D, S575D and N577K) were designed using the RosettaDesign server. The E175N and K576D mutants exhibited improved thermostability in preliminary experiments, so the double mutant E175N/K576D was constructed. These three mutants (E175N, K576D, E175N/K576D) were characterized in detail. The results indicate that the three mutants exhibit a slightly increased optimal temperature (35°C), compared with that of the wild-type enzyme (30°C). The mutants also share an identical pH optimum of 6.0, which is similar to that of the wild-type enzyme. The half-lives of the E175N, K576D and E175N/K576D mutants were 2.30, 1.78 and 7.65 times greater than that of the wild-type enzyme at 45°C, respectively. Kinetic studies showed that the K_m values for the E175N, K576D and E175N/K576D mutants decreased by 6.6%, 2.0% and 11.0%, respectively, and their k_cat/K_m values increased by 38.2%, 4.2% and 19.4%, respectively, compared with those of the wild-type enzyme. After optimizing the conditions for isomaltulose production at 45°C, we found that the E175N, K576D and E175N/K576D mutants displayed slightly improved isomaltulose yields, compared with the wild-type enzyme. Therefore, the mutants produced in this study would be more suitable for industrial biosynthesis of isomaltulose.     

Field performance of a weed-suppressing Serratia plymuthica strain applied with conventional spraying equipment

In previous glasshouse experiments, the soilbacterium Serratia plymuthica, strainA153, showed strong growth-suppressingactivities against a range of broad-leavedweeds after foliar spraying. In field tests ofthis strain in spring wheat, spring barley andpotatoes, variable effects were achieved on arange of weeds including Chenopodiumalbum, Stellaria media, Polygonumconvolvulus and Galeopsis speciosa. Atone site, good suppression of C. albumwas observed when the strain was applied in atank mix with another bacterial isolate or withreduced doses of a herbicide. Effects on weedsappeared to be independent of the applicationvolume (1000, 600, 500 l ha^–1), but weedswere in some cases more strongly suppressed athigher bacterial doses. Barley yields weresomewhat reduced by the bacterial application,but wheat yields were less affected. AlthoughS. plymuthica suppressed certain weedswhen applied in the field in a simple aqueousformulation and with conventional sprayingequipment, the level of weed suppression wasunsatisfactory from a practical standpoint.     

Pleiotropic effects of the chitinase gene from Serratia plymuthica in transgenic potato

The pleiotropic effects of transgenesis includes different consequences of the insertion of a transgene that are not related to the direct action of its product. It is necessary to evaluate the outlook for the application in selection of the transgenic potato strain containing the bacterial chitinase gene chiA we created for studying the possible nonspecific influence of the introduction of the transgene on the phenotypical properties of the transgenic lines. In the present investigation we will consider the effect of the introduction of the chitinase transgene on such agronomically important characteristics as yield and nonspecific resistance.     

Complete genome sequence of the rapeseed plant-growth promoting Serratia plymuthica strain AS9

Serratia plymuthica are plant-associated, plant beneficial species belonging to the family Enterobacteriaceae. The members of the genus Serratia are ubiquitous in nature and their life style varies from endophytic to free-living. S. plymuthica AS9 is of special interest for its ability to inhibit fungal pathogens of rapeseed and to promote plant growth. The genome of S. plymuthica AS9 comprises a 5,442,880 bp long circular chromosome that consists of 4,952 protein-coding genes, 87 tRNA genes and 7 rRNA operons. This genome is part of the project entitled "Genomics of four rapeseed plant growth promoting bacteria with antagonistic effect on plant pathogens" awarded through the 2010 DOE-JGI Community Sequencing Program (CSP2010).     

Draft genome sequence of the antagonistic rhizosphere bacterium Serratia plymuthica strain PRI-2C

Serratia plymuthica strain PRI-2C is a rhizosphere bacterial strain with antagonistic activity against different plant pathogens. Here we present the 5.39-Mb (G+C content, 55.67%) draft genome sequence of S. plymuthica strain PRI-2C with the aim of providing insight into the genomic basis of its antagonistic activity.     

Serratia marcescens forms a new type of cytolysin

Most Serratia marcescens strains produce a new type of cytolysin (hemolysin) which is also found in other Serratia species. The hemolytic polypeptide ShlA (M(r) 162 101) is secreted across the outer membrane through the help of the ShlB protein which also involves conversion of an inactive precursor in an hemolytically active form. Both proteins are synthesized with signal sequences which are released during export across the cytoplasmic membrane. Mutants expressing inactive ShlB derivatives are impaired in activation and secretion suggesting a tight coupling between both processes. The region of ShlA for activation and secretion is confined to the N-terminal 16% of the polypeptide which contains the sequence NPNG which is also found in the Proteus hemolysin, the Bordetella pertussis filamentous hemagglutinin and two highly expressed outer membrane proteins of Haemophilus influenzae. Substitution of the first asparagine (N) residue by isoleucine converts the Serratia hemolysin into an inactive secretion incompetent form. It is concluded that this region is recognized by ShlB for activation and secretion of ShlA. The Serratia hemolysin forms defined pores in erythrocyte membranes.     

Isolation of Plasmid-Harboring Serratia plymuthica from Facultative Gut Microflora of the Tobacco Hornworm, Manduca sexta

Aseptic isolation of the facultative gut microflora of the tobacco hornworm, Manduca sexta, yielded four microorganisms. Two were gram-positive Bacillus spp., one was Serratia plymuthica, and another was the yeast Candida guilliermondii. The three bacterial species were screened for extrachromosomal DNA, and S. plymuthica was found to have a 6.4-kilobase plasmid, which was designated pCP-1.     

Isolation of Shigella dysenteriae type 1 and S. flexneri strains from surface waters in Bangladesh: comparative molecular analysis of environmental Shigella isolates versus clinical strains

Bacillary dysentery caused by Shigella species is a public health problem in developing countries including Bangladesh. Although, shigellae-contaminated food and drinks are often the source of the epidemic's spread, the possible presence of the pathogen and transmission of it through environmental waters have not been adequately examined. We analyzed surface waters collected in Dhaka, Bangladesh, for the presence of shigellae by a combination of PCR assays followed by concentration and culturing of PCR-positive samples. Analysis of 128 water samples by PCR assays for Shigella-specific virulence genes including ipaBCD, ipaH, and stx1 identified 14 (10.9%) samples which were positive for one or more of these virulence genes. Concentration of the PCR-positive samples by filtration followed by culturing identified live Shigella species in 11 of the 14 PCR-positive samples. Analysis of rRNA gene restriction patterns (ribotype) showed that the environmental isolates shared ribotypes with a collection of clinical isolates, but in contrast to the clinical isolates, 10 of the 11 environmental isolates were either negative or carried deletions in the plasmid-encoded invasion-associated genes ipaB, ipaC, and ipaD. However, all environmental Shigella isolates were positive for the chromosomal multicopy invasion-associated gene ipaH and all Shigella dysenteriae type 1 isolates were positive for the stx1 gene in addition to ipaH. This study demonstrated the presence of Shigella in the aquatic environment and dispersion of different virulence genes among these isolates which appear to constitute an environmental reservoir of Shigella-specific virulence genes. Since critical virulence genes in Shigella are carried by plasmids or mobile genetic elements, the environmental gene pool may contribute to an optimum combination of genes, causing the emergence of virulent Shigella strains which is facilitated in particular by close contact of the population with surface waters in Bangladesh.     

Quorum-sensing signaling is required for production of the antibiotic pyrrolnitrin in a rhizospheric biocontrol strain of Serratia plymuthica

One mechanism that bacteria have adopted to regulate the production of antimicrobial compounds is population-density-dependent LuxRI-type quorum sensing (QS), exploiting the production of N-acyl homoserine lactone (AHL) autoinducer signals. In biocontrol bacteria, most known cases involve the AHL control of phenazine antibiotics production by rhizospheric pseudomonads. This work is the first to demonstrate that phenazines are not the only group of biocontrol-related antibiotics whose production is regulated by QS systems. Strain HRO-C48 of Serratia plymuthica isolated from the rhizosphere of oilseed rape and described as a chitinolytic bacterium, which protects crops against Verticillium wilt, was also shown to produce wide-range antibiotic pyrrolnitrin and several AHLs, including N-butanoyl-HSL, N-hexanoyl-HSL and N-3-oxo-hexanoyl-HSL (OHHL). The genes splI and splR, which are analogues of luxI and luxR genes from other Gram-negative bacteria, were cloned and sequenced. The mutant AHL-4 (splI::miniTn5) was simultaneously deficient in the production of AHLs and pyrrolnitrin, as well as in its ability to suppress the growth of several fungal plant pathogens in vitro. However, pyrrolnitrin production could be restored in this mutant by introduction of the splIR genes cloned into a plasmid or by addition of the conditioned medium from strain C48 or OHHL standard to the growth medium.     

Tolerance of Mesorhizobium type strains to different environmental stresses

The symbiosis between rhizobia and legumes is affected by different environmental conditions. Our aims were to evaluate stress tolerance of Mesorhizobium species and investigate species-specific stress response mechanisms. Tolerance of Mesorhizobium type strains to temperature, salt and pH stress was evaluated. Mesorhizobium thiogangeticum showed highest growth with 1.5% NaCl and Mesorhizobium ciceri at pH 5. Mesorhizobium plurifarium showed higher growth at 37°C. SDS-PAGE analysis revealed changes in the protein profiles, namely the overexpression of a 60 kDa protein, following heat stress. Under salt stress, five overexpressed proteins were identified in M. plurifarium and M. thiogangeticum. Northern analysis revealed an increase in groEL expression in Mesorhizobium huakuii and Mesorhizobium septentrionale after heat shock; by contrast, a decrease was detected in Mesorhizobium albiziae and M. thiogangeticum, upon salt shock. A high diversity in tolerance to temperature, salt and pH stress was detected among Mesorhizobium species. M. thiogangeticum and M. ciceri are moderately halophilic and acidophilic, respectively. Several proteins, overproduced in different strains, may be involved in stress tolerance. groEL expression increased upon heat and decreased upon salt shock. To our knowledge, this is the first study focusing tolerance to temperature, salt and pH stress, as well as groEL expression, in Mesorhizobium type strains.     

Overproduction of Serratia marcescens metalloprotease (SMP) from the recombinant Serratia marcescens strains

Summary To overproduce Serratia marcescens metalloprotease(SMP), various recombinant plasmids encoding SMP gene were constructed and the SMP productivities from the recombinant S. marcescens strains were examined. The recombinant S. marcescens strains indispensably required proteinaceous substrates such as casein for the extracellular production of SMP. We obtained maximum 9,100U/ml of SMP from the culture supernatant of S. marcescens ATCC27117 containing a regulatory plasmid pTSP2 encoding SMP gene fused with a strong trc99a promoter and its repressor gene lacI^q, which is about 23 times higher than that of wild type strain. SMP produced from the recombinant S. marcescens(pTSP2) was 88.3% of total extracellular proteins.     

Draft Genome Sequence of the Biocontrol Strain Serratia plymuthica A30, Isolated from Rotting Potato Tuber Tissue

Serratia plymuthica A30 is a Gram-negative bacterium expressing antagonistic activity toward blackleg- and soft rot-causing Dickeya sp. biovar 3 (“Dickeya solani”). Here, we present the draft genome sequence of strain A30, which has been isolated from rotten potato tuber tissue.     

Improvement of seed bio-priming of oilseed rape (Brassica napus ssp. oleifera) with Serratia plymuthica and Pseudomonas chlororaphis

Seed bio-priming of oilseed rape (Brassica napus) with the antagonistic rhizobacteria Serratia plymuthica and Pseudomonas chlororaphis was improved. With the imbibition of water, bacteria are transported into the seed where they survive better. To obtain a minimum bacterial density in the seed of log₁₀ 5 colony-forming-units (CFUs) seed^?1, the bacterial density in the bio-priming suspension should be >log₁₀ 9 CFUs mL^–1 for S. plymuthica and >log₁₀ 8 CFUs mL^–1 for P. chlororaphis. Priming duration was reduced from 12 to 2 h for S. plymuthica and 4 h for P. chlororaphis. Among other priming solutions tested, the addition of MgSO₄ best supported establishment in the seeds and also improved germination. The optimal bio-priming temperature for S. plymuthica is 28°C and for P. chlororaphis 22°C. Survival of the bacteria inside the seeds was moderately improved by storage at low temperature but considerably prolonged by storage under anaerobic conditions. P. chlororaphis survived significantly longer than S. plymuthica.