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+).     

Enterobacter agglomerans: the clinically important plant pathogen.

During a 5-month period Enterobacter agglomerans, now described as a member of the phytopathogenic genus Erwinia, was isolated from 13 patients in a general hospital; in 1 patient it was isolated from two sites. In six instances the organism was the sole pathogen isolated, in two instances it may have contributed to infection and in the remaining instances it was probably a transient saprophyte. The strains showed some variation in biochemical reactions but were similar in colonial morphology and were consistently sensitive to several antibiotics. Although this organism is prevalent in the general environment and usually relatively benign, it does have a potential for nosocomial infection.     

Effects of two locust control methods on wood-eating termites in arid Australia

Termites are ubiquitous detritivores and are a key influence on soil function and nutrient cycles, particularly in arid and semi-arid ecosystems. Locust control presents a unique hazard to termites and the effective functioning of ecosystems as a consequence of the overlap between pesticide applications and termite populations in grassland and desert landscapes. We monitored the effects of locust control methods using ultra-low-volume (ULV) barrier application of a chemical pesticide, fipronil, and a blanket application of a fungal biopesticide, Metarhizium acridum, on wood-eating termites in arid western New South Wales, Australia. We tested the hypothesis that spray applications decrease termite activity at wood baits using a BACI designed field experiment over 2 years. Our replicated control and treatment sites represented the spatial scale of Australian locust control activities. There was no detectable impact of either locust control treatment on termite activity, bait mass loss or termite community composition measures. Non-significant differences in termite survey measures among sites suggested that climate and environmental conditions were stronger drivers of our termite measures than the single, localized and unreplicated application of pesticides more commonly used in locust control operations in arid Australia. A lack of evidence for an impact of our fipronil or Metarhizium application methods supports their use as low hazard locust control options with minimal large scale and longer-term effects on termites in Australian arid rangelands. Future research would be necessary to determine the probable short-term impacts of treatments on individual termite colonies and the possible impacts on non-wood eating termite species in the arid-zone.     

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。     

体内遗传标记成团肠杆菌固氮质粒pEA9

用卡那霉素抗性（Kanr）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3.0 kb片段（nif ENX）克隆到pBR322载体中,再将卡那霉素抗性（Kanr）基因插入到3.0 kb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。 Abstract:The authors describe the in vivo labelling of the plasmid pEA9 in Enterobacter agglomerans 339 with a kanamycin resistance gene.For labelling purposes the donor plasmid pST5 was constructed.This plasmid contains the nif ENX region from pEA9,in which a kanamycin resistance gene is cloned.pST5 was transformed into E.a.339 and subsequently cured from the host.Curing was achieved with AP medium.Fourty strains that had lost pST5,but retained the kanamycin resistance,could be isolated.It showed that none of these clones contained co-integrates of pST5 and pEA9.This is evident that in all clones the kanamycin resistance gene was integrated into pEA9 by homologous recombination.     

Iron reduction in the metal-rich guts of wood-feeding termites

Termites play important roles in lignocellulose and humus turnover in diverse terrestrial ecosystems, and are significant sources of global atmospheric methane and carbon dioxide. All known termite species engage in obligate, complex nutritional symbioses with their gut microbes to carry out such processes. Several hundred microbial species, representing a broad phylogenetic and physiological diversity, are found within the well‐bounded, microliter‐in‐scale gut ecosystem of a given termite. However, most of these species have never been obtained in laboratory culture, and little can be said about their functional roles in the gut community or symbiosis. Herein, an unappreciated facet of the gut chemistry and microbiology of wood‐feeding termites is revealed: the redox metabolism of iron. Gut fluids from field‐collected termites contained millimolar amounts of ferrous iron and other heavy metals. When iron(III) hydroxides were amended to a filter paper diet of Zootermopsis nevadensis, a dampwood termite collected in the San Gabriel Mountains of Southern California, the specimens accumulated high levels of iron(II) in their guts. Additionally, iron was reduced at rapid initial rates in anoxic gut homogenates prepared from field‐collected Z. nevadensis specimens. A Clostridium sp. and a Desulfovibrio sp. were isolated from dilution‐to‐extinction enrichments of Z. nevadensis gut contents and were found to reduce iron(III), as did the termite gut spirochete Treponema primitia. The iron in the guts of wood‐feeding termites may influence the pathways of carbon‐ and electron‐flow, as well as microbial community composition in these tiny ecosystems of global importance.     

六株成团肠杆菌 (Enterobacteragglomerans)接合子的分子生物学分析

对 6株成团肠杆菌 (Enterobacteragglomerans)接合子的分子生物学进行了分析 .6株菌与nifHDK基因有杂交 .菌株总DNA经BamHⅠ酶切后与pEA9 DNA进行Southern杂交 ,只有 2株菌具有完整的质粒DNA ,其余菌株质粒DNA发生了 15 3～ 137 7kb不同程度的缺失 .用切割位点较少的限制性内切酶XbaⅠ酶切 6株菌的总DNA ,经脉冲场凝胶电泳 (PFGE)后用pEA9 DNA为探针进行Southern杂交 ,每株菌的pEA9 DNA明显大于用BamHⅠ酶切后的杂交结果 ,表明质粒与染色体发生了整合 .转座子Tn5或插入序列IS 12 2 2和IS 12 71可能参与质粒与染色体的整合过程 .     

In-situ oxygen profiling and lignin modification in guts of wood-feeding termites

Abstract Reports on the capability of wood-feeding termites (WFTs) in degrading wood particles and on the existence of aerobic environment in the localized guts suggest that their high efficiency of cellulose utilization is not only caused by cellulase, but also by biochemical factors that pretreat lignin. We thus extend the hypothesis that for highly efficient accessibility of cellulose, there should be direct evidence of lignin modification before the hindgut. The lignin degradation/modification is facilitated by the oxygenated environment in intestinal microhabitats. To test our hypothesis, we conducted experiments using a dissolved oxygen microelectrode with a tip diameter < 10 μm to measure oxygen profiles in intestinal microhabitats of both Coptotermes formosanus (Shiraki) and Reticulitermes flavipes (Kollar). Lignin modification during passage through their three gut segments was also analyzed with pyrolysis gas chromatography/mass spectrometry. The data showed relatively high levels of oxygen in the midgut that could have promoted lignin oxidation. Consistent with the oxygen measurements, lignin modifications were also detected. In support of previously proposed hypotheses, these results demonstrate that lignin disruption, which pretreats wood for cellulose utilization, is initiated in the foregut, and continues in the midgut in both termites.     

Heterotrophic bacteria present in hindguts of wood-eating termites [Reticulitermes flavipes (Kollar)]

Strict anaerobic culture techniques were used to quantitate heterotrophic bacteria present in hindguts of Reticulitermes flavipes. The grand mean number of viable cells per hindgut was 0.4 X 10(5) (first-instar larvae), 1.3 X 10(5) (third-instar larvae), 3.5 X 10(5) (workers), and 1.5 X 10(5) (soldiers). Of a total of 344 isolates, 66.3% were streptococci that were always obtained regardless of the origin of termites, their developmental stage or caste, or their length of captivity. Most of the remaining isolates were strains of Bacteroides and Enterobacteriaceae. A small percentage were strains of Lactobacillus, Fusobacterium, and unidentified anaerobic gram-positive rods. Recovery of bacteria from worker hindguts was 13.0% of the direct microscopic count. Isolations performed aerobically failed to reveal strict aerobes. Attempts to isolate cellulolytic bacteria were uniformly unsuccessful. Of 145 streptococcal strains isolated from freshly collected termites, almost all were Streptococcus lactis and S. cremoris. Enterobacteriaceae isolates from the same termite specimens were indole-positive Citrobacter, citrate-negative Citrobacter, and Enterobacter cloacae. The possibility of in situ interspecies lactate transfer, between lactate producers (e.g., streptococci) and lactate fermenters (Bacteroides), is discussed.     

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.     

Salt stress sensitivity of nitrogen fixation in Enterobacter agglomerans strains

Two strains 333 to 339 of Enterobacter agglomerans were selected in the present study to evaluate the response of increasing concentrations of NaCl on growth, N(2)-fixation, and nitrogenase activity/synthesis. E. agglomerans strains 333 and 339 showed optimum growth and acetylene-reducing activity with 0.5 to 1.0% NaCl in a nitrogen-free minimal medium (NFDM) with glucose, respectively, in 28 h incubation, although both strains displayed better growth and acetylene-reducing activity with 3.0% and 2.0% NaCl after 52 h and 100 h incubation periods than the 28 h culture did. Our experiments with shiftings of salt concentrations in NFDM medium indicated that a synthesis of nitrogenase enzyme was generally more sensitive to higher concentrations of NaCl than nitrogenase activity was.     

A comparison of two methods for sampling assemblages of subterranean,wood-eating termites (Isoptera)

Abstract Within a 50 × 50 m area of wandoo Eucalyptus capillosa woodland in the Western Australian wheatbelt, the diversity and frequency of occurrence of wood-eating termite species was assessed at two food types. Over a 12 month period, monthly termite activity was determined: (i) at sound/undecayed artificial baits (seasoned wooden stakes of Jarrah, Karri, Pine, Batu, Oregon; Jarrah sawdust; paper rolls); and (li) at naturally occurring timber, fallen logs and branches of wandoo, in varying stages of decay. Termite diversity was 11 species at baits, 18 species at wandoo out of an overall site richness of 21 species. Karri attracted the most species (9); sawdust attracted none. At wandoo, Nasutitermes exitiosus, Coptotermes acinaciformis and Occasitermes occasus accounted for 59% of samples where termites were recorded. At baits, Heterotermes occiduus accounted for a mean of 80% of samples across bait types, but was rarely sampled at wandoo (5% of samples). Only H. occiduus, C. acinaciformis and Amitermes neogermanus ate bait. Pine, Oregon and paper rolls were most effective in attracting foraging termites in terms of highest per cent of replicates showing bait consumption and highest consumption rates. Jarrah and Batu were least attractive to foraging termites. Samples from wandoo underestimated the relative frequency of occurrence of H. occiduus within the study site. Coptotermes acinaciformis, which attack large food items, and certain species of Amitermes, which forage on subterranean food, may have been underestimated by both sampling methods. These findings indicate that a proper understanding of the structure of wood-eating termite assemblages within a given area requires a composite sampling strategy which addresses termites that eat sound or decayed wood, as well as surface and subsurface foragers.     

Isolation of endophytic diazotroph Pantoea agglomerans and nondiazotroph Enterobacter asburiae from sweetpotato stem in Japan

AIMS: To isolate and identify diazotrophic endophytes in the stem of Japanese sweetpotato cv. Koganesengan. METHODS AND RESULTS: Surface-sterilized and thinly sliced (1-2 mm) sweetpotato stem samples were incubated in test tubes with semi-solid modified Rennie (MR) medium. The test tubes were assayed for acetylene reduction activity (ARA) 5 days after incubation at 30 degrees C. Twelve isolates were obtained from MR plates inoculated with a loop of semi-solid MR medium from ARA+ tubes. However, ARA test showed that only nine isolates were diazotrophic and three were nondiazotrophic strains. Using the API 20E diagnostic kit, four diazotrophic isolates were identified as strains of Pantoea spp. and five isolates as Klebsiella spp. The nondiazotrophic bacteria were strains of Enterobacter spp. A diazotrophic isolate Pantoea sp. MY1 and nondiazotrophic isolate Enterobacter sp. MY2 were identified to the species level by full sequence analysis of 16S rRNA gene. The results showed that MY1 had 99.2% similarity to Pantoea agglomerans ATCC 27155 and MY2 had 99.5% similarity to Enterobacter asburiae ATCC 35953. CONCLUSION: The stem of sweetpotato cv. Koganesengan was colonized by diazotrophic endophyte P. agglomerans and nondiazotrophic endophyte E. asburiae. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is an essential step toward understanding the ecology and interaction between endophytic bacteria and sweetpotato.     

Susceptibility to antibiotics of Enterobacter spp. rods isolated from urine

Enterobacter spp. rods are opportunistic microorganisms which cause of urinary tract infections. The aim of this study was the evaluation of the susceptibility to antimicrobial agents antibiotics of Enterobacter spp. rods isolated from urine. The study was carried 50 of Enterobacter spp strains isolated in the Clinical Microbiology Department of dr. A. Jurasz University Hospital. Antibiotic susceptibility was tested by disk diffusion method. All of strains were susceptible to imipenem and meropenem. There was 87,5% of strains sensitive to doripenem, 79,2% to ertapenem, 54,0% to piperacillin/tazobactam and 50,0% to cephepime. The relatively high percentage (62,0%) of Enterobacter spp. was sensitive to fluoroquinolones. Extended spectrum beta-lactamases were produced by 24 (48,0%) strains.     

Conversion of d-psicose to allitol by Enterobacter agglomerans strain 221e

Enterobacter agglomerans strain 221e transformed d-psicose to allitol at a faster rate in the presence of d-glucose in the reaction mixture when the entrance of oxygen was restricted. Cells grown on glycerol were found to be suitable for the transformation reaction. The transformation rates were about 97.0, 95.0 and 62.5%, respectively, when 0.5, 1.0 and 2.0% substrates were used. No consumption of substrate or product was observed in any case. In flask and jar fermentor reactions, the cells of strain 221e showed similar transformation activities. Cells stored at −20°C for 10 d showed almost same transformation activity as intact cells.     

Partial Purification and Characterization of an Inducible Indole-3-Acetyl-L-Aspartic Acid Hydrolase from Enterobacter agglomerans

Indole-3-acetyl-amino acid conjugate hydrolases are believed to be important in the regulation of indole-3-acetic acid (IAA) metabolism in plants and therefore have potential uses for the alteration of plant IAA metabolism. To isolate bacterial strains exhibiting significant indole-3-acetyl-aspartate (IAA-Asp) hydrolase activity, a sewage sludge inoculation was cultured under conditions in which IAA-Asp served as the sole source of carbon and nitrogen. One isolate, Enterobacter agglomerans, showed hydrolase activity inducible by IAA-L-Asp or N-acetyl-L-Asp but not by IAA, (NH4)2SO4, urea, or indoleacetamide. Among a total of 17 IAA conjugates tested as potential substrates, the enzyme had an exclusively high substrate specificity for IAA-L-Asp. Substrate concentration curves and Lineweaver-Burk plots of the kinetic data showed a Michaelis constant value for IAA-L-Asp of 13.5 mM. The optimal pH for this enzyme was between 8.0 and 8.5. In extraction buffer containing 0.8 mM Mg2+ the hydrolase activity was inhibited to 80% by 1 mM dithiothreitol and to 60% by 1 mm CuSO4; the activity was increased by 40% with 1 mM MnSO4. However, in extraction buffer with no trace elements, the hydrolase activity was inhibited to 50% by either 1 mM dithiothreitol or 1% Triton X-100 (Sigma). These results suggest that disulfide bonding might be essential for enzyme activity. Purification of the hydrolase by hydroxyapatite and TSK-phenyl (HP-Genenchem, South San Francisco, CA) preparative high-performance liquid chromatography yielded a major 45-kD polypeptide as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.