Complete Genome Sequence of the Endophytic Enterobacter cloacae subsp. cloacae Strain ENHKU01

Enterobacter cloacae subsp. cloacae strain ENHKU01 is a Gram-negative endophyte isolated from a diseased pepper (Capsicum annuum) plant in Hong Kong. This is the first complete genome sequence report of a plant-endophytic strain of E. cloacae subsp. cloacae.     

Genomic diversity within the Enterobacter cloacae complex

Background

Isolates of the Enterobacter cloacae complex have been increasingly isolated as nosocomial pathogens, but phenotypic identification of the E. cloacae complex is unreliable and irreproducible. Identification of species based on currently available genotyping tools is already superior to phenotypic identification, but the taxonomy of isolates belonging to this complex is cumbersome.

Methodology/Principal Findings

This study shows that multilocus sequence analysis and comparative genomic hybridization based on a mixed genome array is a powerful method for studying species assignment within the E. cloacae complex. The E. cloacae complex is shown to be evolutionarily divided into two clades that are genetically distinct from each other. The younger first clade is genetically more homogenous, contains the Enterobacter hormaechei species and is the most frequently cultured Enterobacter species in hospitals. The second and older clade consists of several (sub)species that are genetically more heterogonous. Genetic markers were identified that could discriminate between the two clades and cluster 1.

Conclusions/Significance

Based on genomic differences it is concluded that some previously defined (clonal and heterogenic) (sub)species of the E. cloacae complex have to be redefined because of disagreements with known or proposed nomenclature. However, further improved identification of the redefined species will be possible based on novel markers presented here.     

Effects of oxygen stress on chromate reduction in Enterobacter cloacae strain HO1

Enterobacter cloacae strain HO1 was able to reduce toxic hexavalent chromium (chromate) anaerobically. The reduction of chromate by E. cloacae cells was sensitive to oxygen stress. Cultures under continuous aeration showed no chromate reduction. However, when released from the oxygen stress, the cultures readily resumed chromate reduction.     

Cloning and Characterization of Aerobactin Biosynthesis Genes of the Biological Control Agent Enterobacter cloacae

Five strains of Enterobacter cloacae that are biological control agents of Pythium damping-off diseases produced the hydroxamate siderophore aerobactin under iron-limiting conditions. Genes determining aerobactin biosynthesis of the biocontrol strain E. cloacae EcCT-501 were localized to a 12.3-kb region, which conferred aerobactin production to Escherichia coli DH5α. The aerobactin biosynthesis genes of E. cloacae hybridized to those of the pColV-K30 plasmid of E. coli, but restriction patterns of the aerobactin regions of pColV-K30 and E. cloacae differed. A derivative strain with a deletion in the aerobactin biosynthesis locus was as effective as strain EcCT-501 in biological control of Pythium damping-off of cucumber. Thus, aerobactin production did not contribute significantly to the biological control activity of EcCT-501 under the conditions of this study.     

Restriction endonuclease EcaI from Enterobacter cloacae

Restriction endonuclease EcaI obtained from Enterobacter cloacae DSM30056 recognizes the group of heptanucleotide palindromes 5′-G[unk]G-T-N-A-C-C-3′, and on cleavage (arrow) produces fragments with 5′-terminal pentanucleotide extensions. It is identical in specificity with restriction endonuclease BstEII from Bacillus stearothermophilus ET.     

Isolation and Characterization of an Enterobacter cloacae Strain That Reduces Hexavalent Chromium under Anaerobic Conditions

An Enterobacter cloacae strain (HO1) capable of reducing hexavalent chromium (chromate) was isolated from activated sludge. This bacterium was resistant to chromate under both aerobic and anaerobic conditions. Only the anaerobic culture of the E. cloacae isolate showed chromate reduction. In the anaerobic culture, yellow turned white with chromate and the turbidity increased as the reduction proceeded, suggesting that insoluble chromium hydroxide was formed. E. cloacae is likely to utilize toxic chromate as an electron acceptor anaerobically because (i) the anaerobic growth of E. cloacae HO1 accompanied the decrease of toxic chromate in culture medium, (ii) the chromate-reducing activity was rapidly inhibited by oxygen, and (iii) the reduction occurred more rapidly in glycerol- or acetate-grown cells than in glucose-grown cells. The chromate reduction in E. cloacae HO1 was observed at pH 6.0 to 8.5 (optimum pH, 7.0) and at 10 to 40°C (optimum, 30°C).     

Preparation,characterization and immunomodulatory activity of selenium-enriched exopolysaccharide produced by bacterium Enterobacter cloacae Z0206

The tolerant-selenium exopolysaccharide-producing bacterial strain Enterobacter cloacae Z0206 was batch cultured in PDA medium containing optimal concentration of sodium selenite. Selenium was accumulated efficiently in Enterobacter cloacae Z0206 during cultivation with selenium. Inorganic selenite could be transformed into organic forms. Selenium-enriched exopolysaccharide (Se-ECZ-EPS-1) was purified from the fermentation liquid. Selenium content of Se-ECZ-EPS-1 was 12.962 μg/g. Se-ECZ-EPS-1 with Mw of 29,300 Ka was composed of Glc, Gal and Mann with molar ratio of 8.530:0.061:0.706. Administration of Se-ECZ-EPS-1 to cyclophosphamide (CP)-exposed animals resulted in improvement of cellular and humoral immune responses. These findings indicated that Se-ECZ-EPS-1 may act as potent immunomodulatory agents.     

Surface attachment of a sediment isolate ofEnterobacter cloacae

Enterobacter cloacae was recovered from surface sediments of a flood control channel in an area where freshwater runoff mixed with coastal seawater. Cells of this bacterium elaborated an extensive capsule when cultured under laboratory conditions designed to promote extracellular polysaccharide production. Colonization of glass surfaces by cells was similar under aerobic and anaerobic conditions. Temperature exerted little effect on maximum adherent cell density in the range of 15–25°C. The availability of organic nutrients also had little influence on the tendency of cells to adhere to surfaces. Maximum adherent cell densities decreased (76%) as salinity increased from 0 to 12‰ The results suggest that cells ofE. cloacae are suitably adapted to maintain a sessile existence in brackish water sediments of temperate coastal areas.     

Evaluation of the effects of Chlorella vulgaris,Nannochloropsis salina,and Enterobacter cloacae on growth,yield and active compound compositions of Moringa oleifera under salinity stress

Application of Chlorella vulgaris, Nannochloropsis salina and Enterobacter cloacae has been reported to improve the growth of multiple plant species. Moringa oleifera is a medicinal plant found in Saudi Arabia. Its leaves, flowers and fruit have been used as food. Moringa oleifera is rich in rutin and gallic acid and many other bioactive compounds, which collectively contribute to its demonstrated range of pharmacological activities. In Saudi Arabia, the semi-arid and arid weather presents a significant challenge to agriculture. High salinity in cultivated land is a particular threat. We applied Chlorella vulgaris, Nannochloropsis salina, and Enterobacter cloacae at multiple salinities to Moringa oleifera to investigate their effects on the growth, yield, and photosynthetic pigment content. We also examined possible changes in the phytochemical composition. The application of Chlorella vulgaris, Nannochloropsis salina and Enterobacter cloacae enhanced plant growth and yield, while inhibition was observed at high (6000 ppm) salinity. The presence of Chlorella vulgaris and Nannochloropsis salina altered plant growth and yield and rutin and gallic acid content of Moringa oleifera plants grown in saline conditions. Microalgae species were recommended for use as a bio-fertiliser alternative to mainstream synthetic fertilisers.     

AiiA Quorum-Sensing Quenching Controls Proteolytic Activity and Biofilm Formation by Enterobacter cloacae

The aim of this work was to evaluate a quorum-quenching approach to identify functions regulated by quorum sensing in Enterobacter cloacae. We employed an aiiA transconjugant strain of E. cloacae that synthesizes a lactonase enzyme that hydrolyzes N-acyl homoserine lactone signaling molecules to compare bacterial phenotypes in the presence and absence of quorum signals. The aiiA-expressing strain displayed increased proteolytic activity and intensity of a milk-clotting reaction when compared to the wild-type strain. Although both strains growing on polystyrene plates in rich media and a minimal medium of salts formed biofilms, the wild-type strain exhibited a higher number of adhered cells. On the surface of stainless steel coupons that were submerged in culture media, the number of adhered cells of the wild type contained up to one log more cells compared with the aiiA transconjugant. However, after 48?h of incubation, there was no significant difference between the strains. The results demonstrated that the quorum-sensing system negatively regulates proteolytic activity and is likely involved in the early steps of biofilm formation by E. cloacae 067.     

Characterization of the plant growth promoting bacterium,Enterobacter cloacae MSR1, isolated from roots of non-nodulating Medicago sativa

The aim of the present study was to characterize the endophytic bacterial strain designated MSR1 that was isolated from inside the non-nodulating roots of Medicago sativa after surface-sterilization. MSR1 was identified as Enterobacter cloacae using both 16S rDNA gene sequence analysis and API20E biochemical identification system (Biomerieux, France). Furthermore, this bacterium was characterized using API50CH kit (Biomerieux, France) and tested for antibacterial activities against some food borne pathogens. The results showed that E. cloacae consumed certain carbohydrates such as glycerol, d-xylose, d-maltose and esculin melibiose as a sole carbon source and certain amino acids such as arginine, tryptophan ornithine as nitrogen source. Furthermore, MSR1 possessed multiple plant-growth promoting characteristics; phosphate solubility, production of phytohormones acetoin and bioactive compounds. Inoculation of Pisum sativum with MSR1 significantly improved the growth parameters (the length and dry weight) of this economically important grain legume compared to the non-treated plants. To our knowledge, this is the first report addressing E. cloacae which exist in roots of alfalfa growing in Al-Ahsaa region. The results confirmed that E. cloacae exhibited traits for plant growth promoting and could be developed as an eco-friendly biofertilizer for P. sativum and probably for other important plant species in future.     

Aerobic Degradation of 2,4,6-Trinitrotoluene by Enterobacter cloacae PB2 and by Pentaerythritol Tetranitrate Reductase

Enterobacter cloacae PB2 was originally isolated on the basis of its ability to utilize nitrate esters, such as pentaerythritol tetranitrate (PETN) and glycerol trinitrate, as the sole nitrogen source for growth. The enzyme responsible is an NADPH-dependent reductase designated PETN reductase. E. cloacae PB2 was found to be capable of slow aerobic growth with 2,4,6-trinitrotoluene (TNT) as the sole nitrogen source. Dinitrotoluenes were not produced and could not be used as nitrogen sources. Purified PETN reductase was found to reduce TNT to its hydride-Meisenheimer complex, which was further reduced to the dihydride-Meisenheimer complex. Purified PETN reductase and recombinant Escherichia coli expressing PETN reductase were able to liberate nitrogen as nitrite from TNT. The ability to remove nitrogen from TNT suggests that PB2 or recombinant organisms expressing PETN reductase may be useful for bioremediation of TNT-contaminated soil and water.     

Metal Accumulation and Vanadium-Induced Multidrug Resistance by Environmental Isolates of Escherichia hermannii and Enterobacter cloacae

Contaminated soils from an oil refinery were screened for the presence of microorganisms capable of accumulating either nickel, vanadium, or both metals. Three strains of bacteria that belonged to the family Enterobacteriaceae were selected. Two of them were Escherichia hermannii strains, and outer membrane profile (OMP) analysis showed that they were similar to a strain of clinical origin; the other one was an Enterobacter cloacae strain that differed from clinical isolates. The selected bacteria accumulated both nickel and vanadium. Growth in the presence of vanadium induced multidrug resistance phenotypes in E. hermannii and E. cloacae. Incubation with this metal changed the OMP profile of E. hermannii but did not produce variations in the expression of the major OMPs of E. cloacae.     

Enterobacter cloacae A105, isolated from the surface of root nodules of Astragalus sinicus cv. Japan,stimulates nodulation by Rhizobium huakuii bv. renge

Several bacterial strains were isolated from the surface of root nodules of Astragalus sinicus cv. Japan (known as renge-sou in Japanese), a green manure legume that grows in winter and which is used in rice fields to fertilize the soil in both Japan and China. These bacterial strains stimulated the nodulation on renge-sou induced by strains of Rhizobium huakuii bv. renge. From a taxonomic characterization of the isolates, the strains were found to belong to the species Enterobacter cloacae. It was found that strains of E. cloacae increased the number and weight of nodules and the yield of the host plant when these strains were inoculated with a strain of R. huakuii bv. renge both in a test-tube nodulation assay and in soil from a rice field. E. cloacae influenced nodulation at an appropriate ratio of cells of two bacterial strains. The timing of the inoculation of the two strains onto the host plant was also important. The effect of E. cloacae on the nodulation of renge-sou may be due to bacterial products such as exopolysaccharides.     

Differential Inactivation of Seed Exudate Stimulation of Pythium ultimum Sporangium Germination by Enterobacter cloacae Influences Biological Control Efficacy on Different Plant Species

This study was initiated to understand whether differential biological control efficacy of Enterobacter cloacae on various plant species is due to differences in the ability of E. cloacae to inactivate the stimulatory activity of seed exudates to Pythium ultimum sporangium germination. In biological control assays, E. cloacae was effective in controlling Pythium damping-off when placed on the seeds of carrot, cotton, cucumber, lettuce, radish, tomato, and wheat but failed to protect corn and pea from damping-off. Seeds from plants such as corn and pea had high rates of exudation, whereas cotton and cucumber seeds had much lower rates of exudation. Patterns of seed exudation and the release of P. ultimum sporangium germination stimulants varied among the plants tested. Seed exudates of plants such as carrot, corn, lettuce, pea, radish, and wheat were generally more stimulatory to P. ultimum than were the exudates of cotton, cucumber, sunflower, and tomato. However, this was not directly related to the ability of E. cloacae to inactivate the stimulatory activity of the exudate and reduce P. ultimum sporangium germination. In the spermosphere, E. cloacae readily reduced the stimulatory activity of seed exudates from all plant species except corn and pea. Our data have shown that the inability of E. cloacae to protect corn and pea seeds from Pythium damping-off is directly related to its ability to inactivate the stimulatory activity of seed exudates. On all other plants tested, E. cloacae was effective in suppressing damping-off and inactivating the stimulatory activity of seed exudates.     

Fatty Acid Competition as a Mechanism by Which Enterobacter cloacae Suppresses Pythium ultimum Sporangium Germination and Damping-Off

Interactions between plant-associated microorganisms play important roles in suppressing plant diseases and enhancing plant growth and development. While competition between plant-associated bacteria and plant pathogens has long been thought to be an important means of suppressing plant diseases microbiologically, unequivocal evidence supporting such a mechanism has been lacking. We present evidence here that competition for plant-derived unsaturated long-chain fatty acids between the biological control bacterium Enterobacter cloacae and the seed-rotting oomycete, Pythium ultimum, results in disease suppression. Since fatty acids from seeds and roots are required to elicit germination responses of P. ultimum, we generated mutants of E. cloacae to evaluate the role of E. cloacae fatty acid metabolism on the suppression of Pythium sporangium germination and subsequent plant infection. Two mutants of E. cloacae EcCT-501R3, Ec31 (fadB) and EcL1 (fadL), were reduced in β-oxidation and fatty acid uptake, respectively. Both strains failed to metabolize linoleic acid, to inactivate the germination-stimulating activity of cottonseed exudate and linoleic acid, and to suppress Pythium seed rot in cotton seedling bioassays. Subclones containing fadBA or fadL complemented each of these phenotypes in Ec31 and EcL1, respectively. These data provide strong evidence for a competitive exclusion mechanism for the biological control of P. ultimum-incited seed infections by E. cloacae where E. cloacae prevents the germination of P. ultimum sporangia by the efficient metabolism of fatty acid components of seed exudate and thus prevents seed infections.     

New Delhi Metallo-β-Lactamase 1(NDM-1), the Dominant Carbapenemase Detected in Carbapenem-Resistant Enterobacter cloacae from Henan Province,China

The emergence of New Delhi metallo-β-lactamase 1 (NDM-1) has become established as a major public health threat and represents a new challenge in the treatment of infectious diseases. In this study, we report a high incidence and endemic spread of NDM-1-producing carbapenem-resistant Enterobacter cloacae isolates in Henan province, China. Eight (72.7%) out of eleven non-duplicated carbapenem-resistant E. cloacae isolates collected between June 2011 and May 2013 were identified as NDM-1 positive. The bla _NDM-1 gene surrounded by an entire ISAba125 element and a bleomycin resistance gene ble _MBL in these isolates were carried by diverse conjugatable plasmids (IncA/C, IncN, IncHI2 and untypeable) ranging from ~55 to ~360 kb. Molecular epidemiology analysis revealed that three NDM-1-producing E. cloacae belonged to the same multilocus sequence type (ST), ST120, two of which were classified as extensively drug-resistant (XDR) isolates susceptible only to tigecycline and colistin. The two XDR ST120 E. cloacae isolates co-harbored bla _NDM-1, armA and fosA3 genes and could transfer resistance to carbapenems, fosfomycin and aminoglycosides simultaneously via a conjugation experiment. Our study demonstrated NDM-1 was the most prevalent metallo-β-lactamase (MBL) among carbapenem-resistant E.cloacae isolates and identified a potential endemic clone of ST120 in Henan province. These findings highlight the need for enhanced efforts to monitor the further spread of NDM-1 and XDR ST120 E. cloacae in this region.     

Involvement of curli fimbriae in the biofilm formation of Enterobacter cloacae

In this study, we examined the biofilm forming ability, the mRNA expression of curli genes and the morphologies of curli fimbriae and biofilms in clinical isolates of Enterobacter cloacae. The csgBA operon was found in 11 (78.6%) of the 14 isolates. The ability of E. cloacae isolates to form biofilms was significantly correlated with the mRNA expression level of the csgA and csgD genes. The curli protein fimbriae appeared as tangled fibers and the curli-proficient strain formed mature biofilms. Our data suggest that the expression of the curli fimbriae play an important role in biofilm formation in E. cloacae.     

Bacterial reduction of toxic hexavalent chromium using a fed-batch culture of Enterobacter cloacae strain HOl

Bacterial reduction of hexavalent chromium (chromate: CrO₄²⁻) was investigated using fed-batch cultures of Enterobacter cloacae strain HOl. In the fed-batch cultures, toxic CrO₄²⁻ was continuously added in small doses to minimize the toxic effect. The fed-batch process was proved to be an effective biological method for detoxifying CrO₄²⁻ in aqueous solutions.     

Role of pfkA and General Carbohydrate Catabolism in Seed Colonization by Enterobacter cloacae

Enterobacter cloacae A-11 is a transposon mutant of strain 501R3 that was deficient in cucumber spermosphere colonization and in the utilization of certain carbohydrates (D. P. Roberts, C. J. Sheets, and J. S. Hartung, Can. J. Microbiol. 38:1128–1134, 1992). In vitro growth of strain A-11 was reduced or deficient on most carbohydrates that supported growth of strain 501R3 but was unaffected on fructose, glycerol, and all amino acids and organic acids tested. Colonization by strain A-11 was significantly reduced (P ≤ 0.05) for cucumber and radish seeds compared to that of strain 501R3, but colonization of pea, soybean, sunflower, and sweet corn seeds was not reduced. Pea seeds released several orders of magnitude more total carbohydrates and amino acids than cucumber and radish seeds and approximately 4,000-fold more fructose. Fructose was the only carbohydrate detected in the seed exudates which supported wild-type levels of in vitro growth of strain A-11. Soybean, sunflower, and sweet corn seeds also released significantly greater amounts of fructose and total carbohydrates and amino acids than cucumber or radish seeds. The exogenous addition of fructose to cucumber and radish seeds at quantities similar to the total quantity of carbohydrates released from pea seeds over 96 h increased the populations of strain A-11 to levels comparable to those of strain 501R3 in sterile sand. Molecular characterization of strain A-11 indicated that the mini-Tn5 kanamycin transposon was inserted in a region of the genome with significant homology to pfkA, which encodes phosphofructo kinase. A comparison of strain A-11 with Escherichia coli DF456, a known pfkA mutant, indicated that the nutritional loss phenotypes were identical. Furthermore, the pfkA homolog cloned from E. cloacae 501R3 complemented the nutritional loss phenotypes of both E. coli DF456 and E. cloacae A-11 and restored colonization by strain A-11 to near wild-type levels. These genetic and biochemical restoration experiments provide strong evidence that the quantities of reduced carbon sources found in seed exudates and the ability of microbes to use these compounds play important roles in the colonization of the spermosphere.