Dual activity of Serratia marcescens Pt-3 in phosphate-solubilizing and production of antifungal volatiles.

BACKGROUND: Soil fertility decline and pathogen infection are severe issues for crop production all over the world. Microbes as inherent factors in soil were effective in alleviating fertility decrease, promoting plant growth and controlling plant pathogens et al. Thus, screening microbes with fertility improving and pathogen controlling properties is of great importance to humans. RESULTS: Bacteria Pt-3 isolated from tea rhizosphere showed multiple functions in solubilizing insoluble phosphate, promoting plant growth, producing abundant volatile organic compounds (VOCs) and inhibiting the growth of important fungal pathogens in vitro. According to the 16S rRNA phylogenetic and biochemical analysis, Pt-3 was identified to be Serratia marcescens. The solubilizing zone of Pt-3 in the medium of lecithin and Ca3(PO4)2 was 2.1 cm and 1.8 cm respectively. In liquid medium and soil, the concentration of soluble phosphorus reached 343.9 mg.L- 1, and 3.98 mg.kg- 1, and significantly promoted the growth of maize seedling, respectively. Moreover, Pt-3 produced abundant volatiles and greatly inhibited the growth of seven important phytopathogens. The inhibition rate ranged from 75.51 to 100% respectively. Solid phase micro-extraction coupled with gas chromatography tandem mass spectrometry proved that the antifungal volatile was dimethyl disulfide. Dimethyl disulfide can inhibit the germination of Aspergillus flavus, and severely destroy the cell structures under scanning electron microscopy. CONCLUSIONS: S. marcescens Pt-3 with multiple functions will provide novel agent for the production of bioactive fertilizer with P-solubilizing and fungal pathogens control activity.

Identifying the Molecular Targets of an Anti-pathogenic Hydroalcoholic Extract of Punica granatum Peel Against Multidrug-resistant Serratia marcescens.

BACKGROUND: Antibiotic-resistant members of the family Enterobacteriaceae are among the serious threats to human health globally. This study reports the anti-pathogenic activity of Punica granatum peel extract (PGPE) against a multi-drug resistant, beta-lactamase producing member of this family i.e. Serratia marcescens. OBJECTIVE: This study aimed at assessing the anti-pathogenic activity of PGPE against the gramnegative bacterial pathogen S. marcescens and identifying the molecular targets of this extract in the test bacterium. METHODS: Effect of PGPE on S. marcescens growth and quorum sensing (QS)-regulated pigment production was assessed through broth dilution assay. In vivo anti-infective and prophylactic activity of PGPE was assessed employing the nematode worm Caenorhabditis elegans as a model host. Differential gene expression in PGPE-exposed S. marcescens was studied through a whole transcriptome approach. RESULTS: PGPE was able to modulate QS-regulated pigment production in S. marcescens without exerting any heavy growth-inhibitory effect at concentrations as low as ≥2.5 µg/mL. It could attenuate the virulence of the test bacterium towards the worm host by 22-42% (p≤0.01) at even lower concentrations (≥0.5 µg/mL). PGPE also exerted a post-extract effect on S. marcescens. This extract was found to offer prophylactic benefit too, to the host worm, as PGPE-pre-fed worms scored better (34-51%; p≤0.001) survival in face of subsequent bacterial attack. Differential gene expression analysis revealed that PGPE affected the expression of a total of 66 genes in S. marcescens by ≥1.5 fold. CONCLUSION: The anti-virulence effect of PGPE against S. marcescens is multifaceted, affecting stress-response machinery, efflux activity, iron homeostasis, and cellular energetics of this bacterium notably. Among the major molecular targets identified in this study are LPS export transporter permease (LptF), t-RNA pseudouridine synthase (TruB), etc.

N-Acyl-Homoserine Lactone Quorum Sensing Switch from Acidogenesis to Solventogenesis during the Fermentation Process in Serratia marcescens MG1.

N-acyl-homoserine lactone quorum sensing (AHL-QS) has been shown to regulate many physiological behaviors in Serratia marcescens MG1. In the current study, the effects of AHL-QS on the biosynthesis of acid and neutral products by S. marcescens MG1 and its isogenic ∆swrI with or without supplementing exogenous N-hexanoyl-L-homoserine lactone (C6-HSL) were systematically investigated. The results showed that swrI disruption resulted in rapid pH drops from 7.0 to 4.8, which could be restored to wild type by supplementing C6-HSL. Furthermore, fermentation product analysis indicated that ∆swrI could lead to obvious accumulation for acidogenesis products such as lactic acid and succinic acid, especially excess acetic acid (2.27 g/l) produced at the early stage of fermentation, whereas solventogenesis products by ∆swrI appeared to noticeably decrease by an approximate 30% for acetoin during 32-48 h and by an approximate 20% for 2,3-butanediol during 24-40 h, when compared to those by wild type. Interestingly, the excess acetic acid produced could be removed in an AHL-QS-independent manner. Subsequently, quantitative real-time PCR was used to determine the mRNA expression levels of genes responsible for acidogenesis and solventogenesis and showed consistent results with those of product synthesis. Finally, by close examination of promoter regions of the analyzed genes, four putative luxI box-like motifs were found upstream of genes encoding acetyl-CoA synthase, lactate dehydrogenase, α-acetolactate decarboxylase, and Lys-like regulator. The information from this study provides a novel insight into the roles played by AHL-QS in switching from acidogenesis to solventogenesis in S. marcescens MG1.

Genome sequencing and assessment of plant growth-promoting properties of a Serratia marcescens strain isolated from vermicompost.

BACKGROUND: Plant-bacteria associations have been extensively studied for their potential in increasing crop productivity in a sustainable manner. Serratia marcescens is a species of Enterobacteriaceae found in a wide range of environments, including soil. RESULTS: Here we describe the genome sequencing and assessment of plant growth-promoting abilities of S. marcescens UENF-22GI, a strain isolated from mature cattle manure vermicompost. In vitro, S. marcescens UENF-22GI is able to solubilize P and Zn, to produce indole compounds (likely IAA), to colonize hyphae and counter the growth of two phytopathogenic fungi. Inoculation of maize with this strain remarkably increased seedling growth and biomass under greenhouse conditions. The S. marcescens UENF-22GI genome has 5 Mb, assembled in 17 scaffolds comprising 4662 genes (4528 are protein-coding). No plasmids were identified. S. marcescens UENF-22GI is phylogenetically placed within a clade comprised almost exclusively of non-clinical strains. We identified genes and operons that are likely responsible for the interesting plant-growth promoting features that were experimentally described. The S. marcescens UENF-22GI genome harbors a horizontally-transferred genomic island involved in antibiotic production, antibiotic resistance, and anti-phage defense via a novel ADP-ribosyltransferase-like protein and possible modification of DNA by a deazapurine base, which likely contributes to its competitiveness against other bacteria. CONCLUSIONS: Collectively, our results suggest that S. marcescens UENF-22GI is a strong candidate to be used in the enrichment of substrates for plant growth promotion or as part of bioinoculants for agriculture.

Complete genome analysis of Serratia marcescens RSC-14: A plant growth-promoting bacterium that alleviates cadmium stress in host plants.

Serratia marcescens RSC-14 is a Gram-negative bacterium that was previously isolated from the surface-sterilized roots of the Cd-hyperaccumulator Solanum nigrum. The strain stimulates plant growth and alleviates Cd stress in host plants. To investigate the genetic basis for these traits, the complete genome of RSC-14 was obtained by single-molecule real-time sequencing. The genome of S. marcescens RSC-14 comprised a 5.12-Mbp-long circular chromosome containing 4,593 predicted protein-coding genes, 22 rRNA genes, 88 tRNA genes, and 41 pseudogenes. It contained genes with potential functions in plant growth promotion, including genes involved in indole-3-acetic acid (IAA) biosynthesis, acetoin synthesis, and phosphate solubilization. Moreover, annotation using NCBI and Rapid Annotation using Subsystem Technology identified several genes that encode antioxidant enzymes as well as genes involved in antioxidant production, supporting the observed resistance towards heavy metals, such as Cd. The presence of IAA pathway-related genes and oxidative stress-responsive enzyme genes may explain the plant growth-promoting potential and Cd tolerance, respectively. This is the first report of a complete genome sequence of Cd-tolerant S. marcescens and its plant growth promotion pathway. The whole-genome analysis of this strain clarified the genetic basis underlying its phenotypic and biochemical characteristics, underpinning the beneficial interactions between RSC-14 and plants.

Genetic and biochemical characterization of OXA-405, an OXA-48-type extended-spectrum ß-lactamase without significant carbapenemase activity.

The epidemiology of carbapenemases worldwide is showing that OXA-48 variants are becoming the predominant carbapenemase type in Enterobacteriaceae in many countries. However, not all OXA-48 variants possess significant activity toward carbapenems (e.g., OXA-163). Two Serratia marcescens isolates with resistance either to carbapenems or to extended-spectrum cephalosporins were successively recovered from the same patient. A genomic comparison using pulsed-field gel electrophoresis and automated Rep-PCR typing identified a 97.8% similarity between the two isolates. Both strains were resistant to penicillins and first-generation cephalosporins. The first isolate was susceptible to expanded-spectrum cephalosporins, was resistant to carbapenems, and had a significant carbapenemase activity (positive Carba NP test) related to the expression of OXA-48. The second isolate was resistant to expanded-spectrum cephalosporins, was susceptible to carbapenems, and did not express a significant imipenemase activity, (negative for the Carba NP test) despite possessing a blaOXA-48-type gene. Sequencing identified a novel OXA-48-type ß-lactamase, OXA-405, with a four-amino-acid deletion compared to OXA-48. The blaOXA-405 gene was located on a ca. 46-kb plasmid identical to the prototype IncL/M blaOXA-48-carrying plasmid except for a ca. 16.4-kb deletion in the tra operon, leading to the suppression of self-conjugation properties. Biochemical analysis showed that OXA-405 has clavulanic acid-inhibited activity toward expanded-spectrum activity without significant imipenemase activity. This is the first identification of a successive switch of catalytic activity in OXA-48-like ß-lactamases, suggesting their plasticity. Therefore, this report suggests that the first-line screening of carbapenemase producers in Enterobacteriaceae may be based on the biochemical detection of carbapenemase activity in clinical settings.

Isolation and analyses of uranium tolerant Serratia marcescens strains and their utilization for aerobic uranium U(VI) bioadsorption.

Enrichment-based methods targeted at uranium-tolerant populations among the culturable, aerobic, chemo-heterotrophic bacteria from the subsurface soils of Domiasiat (India's largest sandstone-type uranium deposits, containing an average ore grade of 0.1 % U(3)O(8)), indicated a wide occurrence of Serratia marcescens. Five representative S. marcescens isolates were characterized by a polyphasic taxonomic approach. The phylogenetic analyses of 16S rRNA gene sequences showed their relatedness to S. marcescens ATCC 13880 (≥99.4% similarity). Biochemical characteristics and random amplified polymorphic DNA profiles revealed significant differences among the representative isolates and the type strain as well. The minimum inhibitory concentration for uranium U(VI) exhibited by these natural isolates was found to range from 3.5-4.0 mM. On evaluation for their uranyl adsorption properties, it was found that all these isolates were able to remove nearly 90-92% (21-22 mg/L) and 60-70% (285-335 mg/L) of U(VI) on being challenged with 100 µM (23.8 mg/L) and 2 mM (476 mg/L) uranyl nitrate solutions, respectively, at pH 3.5 within 10 min of exposure. his capacity was retained by the isolates even after 24 h of incubation. Viability tests confirmed the tolerance of these isolates to toxic concentrations of soluble uranium U(VI) at pH 3.5. This is among the first studies to report uranium-tolerant aerobic chemoheterotrophs obtained from the pristine uranium ore-bearing site of Domiasiat.

Role of Serratia marcescens 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.

Isolation and characterization of novel strains of Pseudomonas aeruginosa and Serratia marcescens possessing high efficiency to degrade gasoline, kerosene, diesel oil, and lubricating oil.

Bacteria possessing high capacity to degrade gasoline, kerosene, diesel oil, and lubricating oil were screened from several areas of Hokkaido, Japan. Among isolates, two strains, WatG and HokM, which were identified as new strains of Pseudomonas aeruginosa and Serratia marcescens species, respectively, showed relatively high capacity and wide spectrum to degrade the hydrocarbons in gasoline, kerosene, diesel, and lubricating oil. About 90-95% of excess amount of total diesel oil and kerosene added to mineral salts media as a sole carbon source could be degraded by WatG within 2 and 3 weeks, respectively. The same amount of lubricating oil was 60% degraded within 2 weeks. Strain HokM was more capable than WatG in degrading aromatic compounds in gasoline. This strain could also degrade kerosene, diesel, and lubricating oil with a capacity of 50-60%. Thus, these two isolates have potential to be useful for bioremediation of sites highly contaminated with petroleum hydrocarbons.

Introduction of the Serratia marcescens chiA gene into an endophytic Pseudomonas fluorescens for the biocontrol of phytopathogenic fungi.

An endophytic strain of Pseudomonas fluorescens was isolated from micropropagated apple plantlets and introduced into beans (Phaseolus vulgaris) via their root tips. It was shown to be present as an endophyte in the roots at a level of 1.2 x 10(5) CFU/g fresh weight. The gene coding for the major chitinase of Serratia marcescens, chiA, was cloned under the control of the tac promoter into the broad-host-range plasmid pKT240 and the integration vector pJFF350. Pseudomonas fluorescens carrying tacchiA either on the plasmid or integrated into the chromosome is an effective biocontrol agent of the phytopathogenic fungus Rhizoctonia solani on bean seedlings under plant growth chamber conditions.

Expression of a Serratia marcescens chitinase gene in Sinorhizobium fredii USDA191 and Sinorhizobium meliloti RCR2011 impedes soybean and alfalfa nodulation.

A gene encoding chitinase from Serratia marcescens BJL200 was cloned into a broad-host-range vector (pRK415) and mobilized into Sinorhizobium fredii USDA191. Chitinolytic activity was detected in S. fredii USDA191 transconjugants that carried the S. marcescens chiB gene. Chitinase-producing S. fredii USDA191 formed nodules on soybean cultivar McCall. However, there was a delay in nodule formation and a marked decrease in the total number of nodules formed by the chitinase-producing S. fredii in comparison with the wild-type strain. Expression of chitinase in S. meliloti RCR2011 also impeded alfalfa nodulation. Thin-layer chromatography of 14C-labeled Nod factors from chitinase-producing S. fredii USDA191 revealed hydrolysis of lipochitooligosaccharides.

Iron transport systems of Serratia marcescens.

Serratia marcescens W225 expresses an unconventional iron(III) transport system. Uptake of Fe3+ occurs in the absence of an iron(III)-solubilizing siderophore, of an outer membrane receptor protein, and of the TonB and ExbBD proteins involved in outer membrane transport. The three SfuABC proteins found to catalyze iron(III) transport exhibit the typical features of periplasmic binding-protein-dependent systems for transport across the cytoplasmic membrane. In support of these conclusions, the periplasmic SfuA protein bound iron chloride and iron citrate but not ferrichrome, as shown by protection experiments against degradation by added V8 protease. The cloned sfuABC genes conferred upon an Escherichia coli aroB mutant unable to synthesize its own enterochelin siderophore the ability to grow under iron-limiting conditions (in the presence of 0.2 mM 2.2'-dipyridyl). Under extreme iron deficiency (0.4 mM 2.2'-dipyridyl), however, the entry rate of iron across the outer membrane was no longer sufficient for growth. Citrate had to be added in order for iron(III) to be translocated as an iron citrate complex in a FecA- and TonB-dependent manner through the outer membrane and via SfuABC across the cytoplasmic membrane. FecA- and TonB-dependent iron transport across the outer membrane could be clearly correlated with a very low concentration of iron in the medium. Expression of the sfuABC genes in E. coli was controlled by the Fur iron repressor gene. S. marcescens W225 was able to synthesize enterochelin and take up iron(III) enterochelin. It contained an iron(III) aerobactin transport system but lacked aerobactin synthesis. This strain was able to utilize the hydroxamate siderophores ferrichrome, coprogen, ferrioxamine B, rhodotorulic acid, and schizokinen as sole iron sources and grew on iron citrate as well. In contrast to E. coli K-12, S. marcescens could utilize heme. DNA fragments of the E. coli fhuA, iut, exbB, and fur genes hybridized with chromosomal S. marcescens DNA fragments, whereas no hybridization was obtained between S. marcescens chromosomal DNA and E. coli fecA, fhuE, and tonB gene fragments. The presence of multiple iron transport systems was also indicated by the increased synthesis of at least five outer membrane proteins (in the molecular weight range of 72,000 to 87,000) after growth in low-iron media. Serratia liquefaciens and Serratia ficaria produced aerobactin, showing that this siderophore also occurs in the genus Serratia.

Interspecies compatibility of selenoprotein biosynthesis in Enterobacteriaceae.

Several species of Enterobacteriaceae were investigated for their ability to synthesize selenium-containing macromolecules. Seleniated tRNA species as well as seleniated polypeptides were formed by all organisms tested. Two selenopolypeptides could be identified in most of the organisms which correspond to the 80 kDa and 110 kDa subunits of the anaerobically induced formate dehydrogenase isoenzymes of E. coli. In those organisms possessing both isoenzymes, their synthesis was induced in a mutually exclusive manner dependent upon whether nitrate was present during anaerobic growth. The similarity of the 80 kDa selenopolypeptide among the different species was assessed by immunological and genetic analyses. Antibodies raised against the 80 kDa selenopolypeptide from E. coli cross-reacted with an 80 kDa polypeptide in those organisms which exhibited fermentative formate dehydrogenase activity. These organisms also contained genes which hybridised with the fdhF gene from E. coli. In an attempt to identify the signals responsible for incorporation of selenium into the selenopolypeptides in these organisms we cloned a portion of the fdhF gene homologue from Enterobacter aerogenes. The nucleotide sequence of the cloned 723 bp fragment was determined and it was shown to contain an in-frame TGA (stop) codon at the position corresponding to that present in the E. coli gene. This fragment was able to direct incorporation of selenocysteine when expressed in the heterologous host, E. coli. Moreover, the E. coli fdhF gene was expressed in Salmonella typhimurium, Serratia marcescens and Proteus mirabilis, indicating a high degree of conservation of the seleniating system throughout the enterobacteria.