Direct detection of nif-gene sequences of Enterobacter agglomerans in soil

Abstract: Specific nif sequences of Enterobacter agglomerans plasmid pEA9 were detected in total DNA recovered from soil 70 days after its inoculation with the bacteria, when these were no longer culturable on agar medium. For this, a modified method of DNA extraction from soil was used. No amplification of DNA sequences by PCR was necessary.     

Persistence and stability of genetically manipulated derivatives of Enterobacter agglomerans in soil microcosms

Abstract Molecular methods and conventional plating were applied to monitor Enterobacter agglomerans 339 derivatives carrying a Tn5-Mob or an npt I-cassette in unsterile soil microcosms. The plate counts of the introduced bacteria decreased continuously in time until undetectable on selective media. In contrast, hybridization of the total DNA directly isolated from inoculated soil samples showed that the target sequences detected corresponded to a much higher number of bacteria than indicated by plating. By PCR-amplification and hybridization of the soil DNA we could show that asignificant number of target sequences still persisted in the soil microcosms, even when the inoculated bacteria were not able to make colonies on selective agar plates. The Tn 5 marker caused instabilities in the genome of the bacteria studied. Some of the clones that grew in the soil samples had rearrangements in their genome. The detection of E. agglomerans 339 derivatives carrying the immobile npt I-cassette was also dependent on its location in the bacterial genome.     

Ecological behavior of plasmids and resistance to lead of Enterobacter agglomerans isolated from soil

Soil samples taken monthly in 1990 from five parks outside Tokyo were examined for Enterobacter agglomerans. A total of 348 strains were isolated and 250 of them were tested for the presence of plasmids by DNA agarose gel electrophoresis. All the isolates carried at least two kinds of plasmids. Those isolated from July to September showed four or five kinds of plasmids (group I) and those isolated from January to June and from October to December showed two or three kinds of plasmids (group II). A majority of the plasmids detected were of 1,500 or fewer base pairs. The isolates were tested for the Pb2+ resistance; group I strains were more resistant to lead than group II strains. It is presumed that bacterial plasmids are related with the ecosystem of soil and the resistance to lead in E. agglomerans.     

Purification and partial characterization of azoreductase from Enterobacter agglomerans

Azoreductase, an enzyme catalyzing the reductive cleavage of the azo bond of methyl red (MR) and related dyes, was purified to electrophoretic homogeneity from Enterobacter agglomerans. This bacterial strain, isolated from dye-contaminated sludge, has a higher ability to grow, under aerobic conditions, on culture medium containing 100mg/L of MR. The enzyme was purified approximately 90-fold with 20% yield by ammonium sulfate precipitation, followed by three steps of column chromatography (gel-filtration, anion-exchange, and dye-affinity). The purified enzyme is a monomer with a molecular weight of 28,000 Da. The maximal azoreductase activity was observed at pH 7.0 and at 35 degrees C. This activity was NADH dependent. The K(m) values for both NADH and MR were 58.9 and 29.4 microM, respectively. The maximal velocity (V(max)) was 9.2 micromol of NADH min(-1)mg(-1). The purified enzyme is inhibited by several metal ions including Fe(2+) and Cd(2+).     

Genome sequence of Pantoea agglomerans strain IG1

Pantoea agglomerans is a gram-negative bacterium that grows symbiotically with various plants. Here we report the 4.8-Mb genome sequence of P. agglomerans strain IG1. The lipopolysaccharides derived from P. agglomerans IG1 have been shown to be effective in the prevention of various diseases, such as bacterial or viral infection, lifestyle-related diseases. This genome sequence represents a substantial step toward the elucidation of pathways for production of lipopolysaccharides.     

NonepidemicErwinia herbicola (Enterobacter agglomerans) in blood cultures: Bacteriological analysis of fifteen cases

The finding ofErwinia herbicola (Enterobacter agglomerans) in blood cultures of 15 inpatients, during a 53/4-year period in a nonepidemic context, was analyzed for clinical significance. Mixed cultures were seen in 5 patients; multiple sets were taken in 10 patients. In only 6 patients couldErwinia be significantly correlated with clinical septicemia; in 9, it was of doubtful significance, more likely reflecting transient bacteremia than skin contamination or laboratory handling. These findings contrast to previous reports that stress a preponderance of clinically significant strains ofEnterobacter species andErwinia from blood cultures. The only bacteriological differences between the “significant” and “doubtful” groups involved the time required to detectErwinia.     

体内遗传标记成团肠杆菌固氮质粒pEA9

用卡那霉素抗性（Kan^r)基因对成团肠杆菌固氮质粒pEA9进行活体遗传标记。将来自质粒pEA9的3.0kb片段(nif ENX)克隆到pBR322载体中,再将卡那霉素抗性(Kan^r)基因插入到3.0kb的片段中,构建成供体质粒pST5。将该质粒转化到含有待标记质粒pEA9的E.a.339菌株中,然后在AP培养基中消除供体质粒,筛选得到40个失去了pST5并保持卡那霉素抗性的克隆,分析表明它们不是质粒pEA9和pST5的共整合体,而是卡那霉素抗性基因通过两个质粒在nifENX区域内的DNA间的同源重组整合到了质粒pEA9上。     

Conjugative plasmids in multi-resistant bacterial isolates from Indian soil

Aims:  Determination of heavy metal and antibiotic resistance and presence of conjugative plasmids in bacteria isolated from soil irrigated with wastewater.
Methods and Results:  Composite soil samples were collected from Ghaziabad, Uttar Pradesh, India. Forty different bacteria were selected from nutrient agar and characterized by morphological, cultural and biochemical tests. All the isolates were tested for their resistance to different heavy metals and antibiotics. The DNA derived from multiple metal and antibiotic-resistant bacterial isolates was PCR amplified and plasmid-specific sequences (IncP, IncN, IncW, IncQ and pMV158-type) were analysed by dot blot hybridization. All isolates gave PCR products with trfA2 and oriT primers of the IncP group. These PCR products also hybridized with the RP4-derived probes. However, the samples were negative for all the other investigated plasmids as proved by PCR and dot blots.
Conclusions:  The presence of conjugative/mobilizable IncP plasmids in the isolates indicates that these bacteria have gene-mobilizing capacity with implications for potential dissemination of introduced recombinant DNA.
Significance and Impact of the Study:  The detection of IncP plasmids in all the bacterial isolates is another proof for the prevalence of these plasmids. We propose that IncP plasmids are mainly responsible for the spread of multi-resistant bacteria in these soils.     

Genome trees constructed using five different approaches suggest new major bacterial clades

Background  

The availability of multiple complete genome sequences from diverse taxa prompts the development of new phylogenetic approaches, which attempt to incorporate information derived from comparative analysis of complete gene sets or large subsets thereof. Such attempts are particularly relevant because of the major role of horizontal gene transfer and lineage-specific gene loss, at least in the evolution of prokaryotes.     

Site-specific mutadenesis in Enterobacter agglomerans: construction of nifB mutants and analysis of the gene's structure and function

A novel technique was developed which may be generally well suited to the site-specific construction of mutations in Enterobacter agglomerans. The method is based on the observation that E. agglomerans can be cured of a plasmid of the incompatibility group IncQ by cultivation on citrate-containing medium. To test the applicability of this technique, we inserted a kanamycin cassette into the cloned nifB gene, transferred it into E. agglomerans, and selected for recombinants in which the wild-type nifB was replaced by the mutated gene by growing transformants on citrate medium with kanamycin. The nifB ^? mutants with the kanamycin cassette inserted in either orientation showed sequence of nifb. A typical σ⁵⁴-dependent promoter and a consensus NifA binding site were found upstream of nifB. Activation of this promoter by both heterologous and homologous NifA proteins was observed in vivo. The predicted amino acid sequence of the NifB protein showed strong similarity to the NifB sequences of other diazotrophic bacteria. The typical clustering of cysteine residues at the N-terminal end indicates its involvement in Fe-Mo cofactor biosynthesis.     

Cytotoxic activity of Enterobacter cloacae human isolates

We examined 55 Enterobacter cloacae isolates from clinical specimens for the production of cytotonic and cytotoxic toxins and the presence of the type III secretion system (TTSS). Twelve isolates (22%) revealed cytotoxic activity that caused destruction of Vero cells, whereas 28 (51%) strains induced lysis of the murine macrophage J774 cell line. TTSS genes were present in 27% of the isolates. The results indicated that these bacteria may destroy phagocytes and epithelial cells, which may lead to spread within the host.     

High prevalence of biofilm synergy among bacterial soil isolates in cocultures indicates bacterial interspecific cooperation

Biofilms that form on roots, litter and soil particles typically contain multiple bacterial species. Currently, little is known about multispecies biofilm interactions and few studies have been based on environmental isolates. Here, the prevalence of synergistic effects in biofilm formation among seven different soil isolates, cocultured in combinations of four species, was investigated. We observed greater biofilm biomass production in 63% of the four-species culture combinations tested than in biofilm formed by single-species cultures, demonstrating a high prevalence of synergism in multispecies biofilm formation. One four-species consortium, composed of Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans and Paenibacillus amylolyticus, exhibited strong synergy in biofilm formation and was selected for further study. Of the four strains, X. retroflexus was the only one capable of forming abundant biofilm in isolation, under the in vitro conditions investigated. In accordance, strain-specific quantitative PCR revealed that X. retroflexus was predominant within the four-species consortium (>97% of total biofilm cell number). Despite low relative abundance of all the remaining strains, all were indispensable for the strong synergistic effect to occur within the four-species biofilm. Moreover, absolute individual strain cell numbers were significantly enhanced when compared with those of single-species biofilms, indicating that all the individual strains benefit from inclusion in the multispecies community. Our results show a high prevalence of synergy in biofilm formation in multispecies consortia isolated from a natural bacterial habitat and suggest that interspecific cooperation occurs.     

Molecular cloning, structural analysis, and expression in Escherichia coli of a chitinase gene from Enterobacter agglomerans.

The gene chiA, which codes for endochitinase, was cloned from a soilborne Enterobacter agglomerans. Its complete sequence was determined, and the deduced amino acid sequence of the enzyme designated Chia_Entag yielded an open reading frame coding for 562 amino acids of a 61-kDa precursor protein with a putative leader peptide at its N terminus. The nucleotide and polypeptide sequences of Chia_Entag showed 86.8 and 87.7% identity with the corresponding gene and enzyme, Chia_Serma, of Serratia marcescens, respectively. Homology modeling of Chia_Entag's three-dimensional structure demonstrated that most amino acid substitutions are at solvent-accessible sites. Escherichia coli JM109 carrying the E. agglomerans chiA gene produced and secreted Chia_Entag. The antifungal activity of the secreted endochitinase was demonstrated in vitro by inhibition of Fusarium oxysporum spore germination. The transformed strain inhibited Rhizoctonia solani growth on plates and the root rot disease caused by this fungus in cotton seedlings under greenhouse conditions.     

Characterization of ferrioxamine E as the principal siderophore ofErwinia herbicola (Enterobacter agglomerans)

Summary Several strains of the enterobacterial groupErwinia herbicola (Enterobacter agglomerans) were screened for siderophore production. After 3 days of growth in a low-iron medium, all strains studied produced hydroxamate siderophores. The retention values of the main siderophore during thin-layer chromatography on silica gel plates and on HPLC reversed-phase columns were identical with those of an authentic sample of ferrioxamine E (norcardamine). Gas-chromatographic analysis of the HI hydrolyzate yielded succinic acid and 1,5-diaminopentane in equimolar amounts; fast-atom-bombardment (FAB) mass spectroscopy showed a molecular mass of 653 Da. Iron from⁵⁵Fe-labelled ferrioxamine E was well taken up by iron-starved cells ofE. herbicola (K _m=0.1 M,V _max=8 pmol mg^–1 min^–1). However, besides ferrioxamine E (100%), several exogenous siderophores such as enterobactin (94.5%), ferric citrate (78.5%), coprogen (63.5%) and ferrichrome (17.5%) served as siderophores, suggesting the presence of multiple siderophore receptors in the outer membrane ofE. herbicola.     

Antagonistic and antimicrobial activities of some bacterial isolates collected from soil samples

Thirty seven bacterial cultures isolated from soil samples obtained from different locations were tested for their antagonistic activity against some fungal pathogens, viz., Sclerotium rolfsii, Fusarium oxysporum and Rhizoctonia solani, causal agents of collar rot of sunflower, wilts and root rots, respectively. Among them, 5 bacterial strains, viz., A1 6 (Bacillus sphaericus), K1 24 (Pseudomonas fluorescens), M1 42 (Bacillus circulans), M1 66 (Bacillus brevis) and T1 22 (Bacillus brevis) showed positive antagonistic activity. M1 66 was the most effective in inhibiting mycelial growth of S. rolfsii in vitro followed by M1 42, T1 22, K1 24 and A1 6. Only one bacterial strain i.e. M1 42 exhibited antagonistic activity against F. oxysporum, and none of the bacterial strains gave positive activity against R. solani. Furthermore, antimicrobial activities of all the 5 strains were checked against different test organisms. These strains showed their extensive inhibition effect particularly against gram-positive test bacteria (Staphylococcus aureus and Bacillus subtilis) and the test fungal strain (Candida albicans). On the other hand, B. brevis M1 66 and B. brevis T1 22 strains had an inhibitory effect against gram positive and gram-negative test bacteria (Escherichia coli and Proteus vulgaris) as well as the test fungal strain.     

N-carbobenzyloxy-D-aspartic acid as a competitive inhibitor of indole-3-acetyl-L-aspartic acid hydrolase of Enterobacter agglomerans

The gene for a specific IAA-asp hydrolase from Enterobacteragglomerans, iaaspH, is a potentially useful tool for modificationofIAA homeostasis in higher plants that use the IAA-asp oxidation pathway forauxin catabolism. In order to optimize the utility of this gene for plantmodification and to increase the success of obtainingiaaspH transformed plants from culture, we haveinvestigated aspects of IAA-asp hydrolase catalysis. The catalyticcharacteristics of the IAA-asp hydrolase from Enterobacteragglomerans was studied using ten compounds that are structuralanalogues of IAA-asp. These compounds were tested as potential IAA-asphydrolasesubstrates as well as for inhibition of IAA-asp hydrolysis. Among them,N-carbobenzyloxy-D-aspartic acid (N-CBZ-D-asp) and N-CBZ-L-asp were found to bethe strongest inhibitors with more than 80% inhibition of IAA-asp hydrolysis.Aspartyl-L-aspartic acid and a asp-ser-asp-pro-arg peptide also showed stronginhibitory activities, reducing rates of IAA-L-asp hydrolysis, when added atequal molar amounts relative to the substrate, by 60% and 65%, respectively.N-CBZ-D-asp was chosen for further kinetic studies and for studies of itstoxicity in relation to seed germination because it was a strong inhibitor,exhibited a very low rate of hydrolysis by the IAA-asp hydrolase and wascommercially available. N-CBZ-D-asp was shown to be a competitive inhibitor forthe Enterobacter agglomerans IAA-asp hydrolase with aK_i value of 1.22 mM. Studies oftomato seed germination showed that N-CBZ-D-asp did not affect the rate of seedgermination at up to 1 mM, but the growth rate of seedlings wassignificantly reduced when the concentration in the medium was 0.5mM and higher. These results indicate that, at suitableconcentrations, N-CBZ-D-asp should be a useful tool for control of low levelexpression of the iaaspH in transgenic plants duringcritical stages of plant regeneration from culture.