Quantitative studies on phosphorus transference occuring between <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> and its attached bacterium (<Emphasis Type="Italic">Pseudomonas</Emphasis> sp.)

Phosphorus release from Microcystis aeruginosa and attached bacterium (Pseudomonas sp.) isolated from Lake Taihu was examined using a phosphorus isotope tracer in order to investigate the phosphorus transference between the two species. Our results reveal that the amount of phosphorus released form ³²P-saturated M. aeruginosa is determined by its growth phase and most of phosphorus is assimilated by Pseudomonas finally while the amount of phosphorus released from ³²P-saturated Pseudomonas is also determined by the growth phase of M. aeruginosa and most of them are assimilated by M. aeruginosa. The results suggest that phosphorus transference occurs between M. aeruginosa and its attached Pseudomonas . This process makes microenvironment of mucilage of M. aeruginosa attached bacteria maintain relative high amounts of phosphorus. Attached bacteria may be a temporary phosphorus bank to the growth of M. aeruginosa, and assimilation of phosphorus by M. aeruginosa becomes easy when M. aeruginosa is in lag growth phase. Thus, the phosphorus exchange between M. aeruginosa and attached Pseudomonas in microenvironment may be important to microfood web and cyanobacteria bloom.     

Impact of <Emphasis Type="Italic">cry1AC</Emphasis>-carrying <Emphasis Type="Italic">Brassica rapa</Emphasis> subsp. <Emphasis Type="Italic">pekinensis</Emphasis> on leaf bacterial community

The effects of Chinese cabbage (Brassica rapa subsp. pekinensis) carrying cry1AC derived from Bacillus thuringiensis (Bt) on leaf bacterial community were examined by analyzing the horizontal transfer of trans-gene fragments from plants to bacteria. The effect of plant pathogenic bacteria on the gene transfer was also examined using Pseudomonas syringae pathovar. maculicola. The frequency of hygromycin-resistant bacteria did not alter in Bt leaves, though slight increase was observed in Pseudomonas-infected Bt leaves with no statistical significance. The analysis of bacterial community profiles using the denaturing gradient gel electrophoresis (DGGE) fingerprinting indicated that there were slight differences between Bt and control Chinese cabbage, and also that infected tissues were dominated by P. syringae pv. maculicola. However, the cultured bacterial pools were not found to contain any transgene fragments. Thus, no direct evidence of immediate gene transfer from plant to bacteria or acquisition of hygromycin resistance could be observed. Still, long-term monitoring on the possibility of gene transfer is necessary to correctly assess the environmental effects of the Bt crop on bacteria.     

Cell hydrophobicity of <Emphasis Type="Italic">Pseudomonas</Emphasis> spp. and <Emphasis Type="Italic">Bacillus</Emphasis> spp. bacteria and hydrocarbon biodegradation in the presence of Quillaya saponin

Biodegradation and hydrophobicity of Pseudomonas spp. and Bacillus spp. strains were tested at different concentrations of the biosurfactant Quillaya saponin. A model mixture of hydrocarbon (dodecane and hexadecane) was used for estimating the influence of surfactants on biodegradation. The bacterial adhesion to hydrocarbon method for determination of bacterial cell surface hydrophobicity was exploited. Among the tested bacterial strains the higher hydrophobicity was noticed for Pseudomonas aeruginosa TK. The hydrophobicity of this strain was 84%. The highest hydrocarbon biodegradation was observed for P. aeruginosa TK (49%) and Bacillus subtilis (35%) strains after 7 days of experiments. Generally the addition of Quillaya saponin increased hydrocarbon biodegradation remarkably. The optimal concentration proved to be 80 mg l⁻¹. The degree of hydrocarbon biodegradation was 75% for P. aeruginosa TK after the addition of saponin. However the most significant increase in biodegradation after addition of Quillaya saponin was in the case of P. aeruginosa 25 and Pseudomonas putida (the increase of biodegradation from 21 to 52% and from 31 to 66%, respectively). It is worth mentioning that decrease of hydrophobicity is correlated with the best biodegradation by P. aeruginosa strain. For the remaining strains, no significant hydrophobicity changes in relation to the system without surfactant were noticed.     

Transfer of the chromosomally encoded tetracycline resistance gene <Emphasis Type="Italic">tet</Emphasis>(M) from marine bacteria to <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Enterococcus faecalis</Emphasis>

The transferability of the tetracycline (TC) resistance gene tet(M) from marine bacteria to human enteric bacteria was examined by a filter-mating method. Vibrio spp., Lactococcus garvieae, Bacillus spp., Lactobacillus sp., and Paenibacillus sp. were used as donors, and Escherichia coli JM109 and Enterococcus faecalis JH2-2 were used as recipients. The combination of Vibrio spp. and E. coli resulted in 5/68 positive transconjugants with a transfer rate of 10⁻⁷ to 10⁻³; however, no transfer was observed with E. faecalis. In case of L. garvieae and E. faecalis, 6/6 positive transconjugants were obtained with a transfer rate of 10⁻⁶ to 10⁻⁵; however, no transfer was observed with E. coli. The tet(M) gene of Bacillus, Lactobacillus, and Paenibacillus were not transferred to either E. coli or E. faecalis. tet(M) transfer was confirmed in positive E. coli and E. faecalis transconjugants by polymerase chain reaction (PCR) and Southern hybridization. All the donor strains did not harbor plasmids, while they all harbored transposon Tn916. In the transconjugants, the transposon was not detected by PCR, suggesting the possible transfer of tet(M) from the marine bacterial chromosome to the recipient chromosome. This is the first report to show that tet(M) can be transferred from marine bacteria to human enteric bacteria in a species-specific manner.     

Isolation of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Specific Phages with Broad Activity Spectra

The aim of the study was to screen various kinds of samples for Pseudomonas aeruginosa specific phages and to isolate and partially characterize those with broad activity spectra. The Pseudomonas specific phages were isolated using an enrichment procedure with single strains or the cocktail of P. aeruginosa strains as hosts. Using the described procedure, phages were successfully isolated only from water samples, while in soil and feces no Pseudomonas specific phages were detected. The lytic spectra of isolated phages were determined by spot method on lawns of 33 P. aeruginosa strains and five species belonging to family Enterobacteriaceae. The results showed that among isolated phages, 001A, δ, and I possessed the broad activity spectra, as were able to plaque on more than 50% of tested P. aeruginosa strains, while none of the phages were able to lyse the other tested species. Significant differences in phage activity spectra were not observed when P. aeruginosa cocktail was applied for sample enrichment. The most of the phages examined by electron microscopy belonged to family Siphoviridae, while the broad activity spectra isolates, except for 001A, possessed morphological characteristics of family Podoviridae. Digested DNA of the phages δ and I showed similar patterns, indicating the prevalence and success of this phage type in the environment.     

Copper Uptake by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Isolated from Infected Burn Patients

Pseudomonas aeruginosa was isolated from infected burn patients and characterized by standard biochemical tests. The in vitro copper uptake was compared between this isolated pathogenic strain and two non-pathogenic control strains of Gram positive bacteria Bacillus thuringiensis strain Israelis as well as Gram negative bacteria Enterobacter aerogenes. Maximum copper uptake of 470 ppm/g biomass was obtained by P. aeruginosa strain, while the control strains B. thuringiensis and Enterobacter aerogenes had copper uptake of 350 and 383 ppm/g biomass, respectively. However, the lowest copper uptake (60 ppm/g biomass) was observed with another control the saprophytic strain Pseudomonas (Shewanella) putrefaciens. A further investigation regarding the effect of copper toxicity on bacterial growth, gave an MIC score of 600 ppm for P. aeruginosa strain compared to 460 and 300 ppm for the two Gram positive and Gram negative control strains, respectively. In tandem with these in vitro findings, blood analysis on burn patients infected with P. aeruginosa has indicated a selective decrease of copper (hypocupremia) and ceruloplasmin plasma levels. The iron metabolism was also affected by this copper deprivation leading to a similar decrease in plasma levels of PCV, iron, total iron binding capacity, and transferrin. All these hematological changes were significantly different (P < 0.05) from the matched group of non-infected burn patients. The observed hypocupremia in infected burn patients was attributed to demanding scavenger ability by P. aeruginosa strain for the copper of plasma.     

Rhamnolipid-producing thermophilic bacteria of species <Emphasis Type="Italic">Thermus</Emphasis> and <Emphasis Type="Italic">Meiothermus</Emphasis>

Novel rhamnolipid-producing strains of three thermophilic bacteria, Thermus sp., T. aquaticus and Meiothermus ruber were identified that have not been previously described as rhamnolipid producers. Rhamnolipids were extracted from supernatant and further purified by thin-layer chromatography. Mass spectrometry with negative electrospray ionization revealed 77 rhamnolipid homologues varying in chain length and unsaturation. Tandem mass spectrometry identified mono-rhamnolipid and di-rhamnolipid homologues containing one or two 3-hydroxy-fatty acids, saturated, monounsaturated or diunsaturated, even- or odd-chain, up to unusual long chains with 24 carbon atoms. The stereochemistry of rhamnose was L and that of 3-hydroxy-fatty acids was R, the position of double bonds in monoenoic acids was cis ω-9. All three strains produced a rhamnolipid that differs in structure from Pseudomonas aeruginosa rhamnolipids and exhibits excellent surfactant properties. Importantly, in comparison to P. aeruginosa both strains, i.e., Thermus and Meiothermus, are Biosafety level 1 microorganisms and are not pathogenic to humans.     

Effect of mixed diets (cyanobacteria and green algae) on the population growth of the cladocerans <Emphasis Type="Italic">Ceriodaphnia dubia</Emphasis> and <Emphasis Type="Italic">Moina macrocopa</Emphasis>

Microcystis aeruginosa, a cosmopolitan form, is a colonial cyanobacterium, which is also common in many freshwater bodies in Mexico. In eutrophic water bodies cyanobacteria are often the main phytoplankton that co-exist with cladocerans. We evaluated the effect of mixed diets, comprising 0, 25, 50, 75, and 100% on dry weight basis of M. aeruginosa, and the rest of one of two green algal species (Chlorella vulgaris and Scenedesmus acutus), on the population growth of the cladocerans Ceriodaphnia dubia and Moina macrocopa. Regardless of the share of M. aeruginosa in the mixed diet, C. dubia fed Chlorella had a longer initial lag phase. However, in mixed diet with S. acutus, the lag phase of C. dubia increased with increasing proportion of M. aeruginosa. When raised on 100% M. aeruginosa, the population growth of C. dubia was lowered compared with 100% S. acutus or 100% C. vulgaris. Increased proportion of M. aeruginosa in the mixed diet also resulted in decreased abundance of M. macrocopa. Irrespective of diet type, M. macrocopa had a shorter lag phase than C. dubia. Depending on the diet type, the rate of population increase (r) of C. dubia varied from 0.07 to 0.26 d⁻¹ while that of M. macrocopa was higher (0.14–0.61 d⁻¹). For both cladoceran species, the lower r values were obtained when fed Microcystis. Our study showed that the strain of M. aeruginosa was not highly toxic to cause total elimination of either C. dubia or M. macrocopa. Addition of a green algal component to the diet improved the population growth rates of both cladoceran species.     

<Emphasis Type="Italic">Humulus japonicus</Emphasis> Accelerates the Decomposition of <Emphasis Type="Italic">Miscanthus sacchariflorus</Emphasis> and <Emphasis Type="Italic">Phragmites australis</Emphasis> in a Floodplain

Humulus japonicus in communities of Miscanthus sacchariflorus and Phragmites australis can grow large enough to overtop other species in the Amsa-dong floodplain. Because of strong winds and the weight of Humulus, plants of M. sacchariflorus and P. australis fell in mid-August and were subject to decomposition under its dense shading. To assess the effects of H. japonicus on nutrient cycling in these communities, we collected fresh samples of M. sacchariflorus and P. australis in litterbags and decomposed them under H. japonicus for 9 months, beginning in August. Biomass and organic contents from M. sacchariflorus during this incubation period were 49–51% and 44–48%, whereas those of P. australis were 49–61% and 32–52%, respectively. Their annual k values were 1.61–1.74 and 1.46–3.54, respectively. Initial N concentrations in M. sacchariflorus and P. australis were 13 and 20 mg g⁻¹, while C:N ratios were 31 and 21, respectively. These results indicate that H. japonicus is responsible for the collapse of M. sacchariflorus and P. australis in August and also accelerates their nutrient cycling through rapid decomposition, thereby increasing nutrient circulation in floodplains.     

Structure and expression of gene <Emphasis Type="Italic">vfr</Emphasis> in <Emphasis Type="Italic">Pseudomonas chlororaphis</Emphasis> 449

Gene vfr of Pseudomonas chlororaphis 449 previously described only in Pseudomonas aeruginosa was identified, cloned, and sequenced; its localization in the chromosome was determined. Amino acid sequence of the protein encoded by gene vfr in P. chlororaphis 449 was shown to have a 83% identity with the Vfr protein of P. aeruginosa PAO1 and a 63% identity with the CRP protein of Escherichia coli. Amino acid residues that ensure the most important structural properties of the CRP protein, i.e., its binding to cAMP, RNA polymerase, and DNA, were identical or highly conserved in Vfr proteins of P. aeruginosa and P. chlororaphis 449. The cloned vfr gene of P. chlororaphis 449 was complemented partially the mutation at gene crp in cells of E. coli AM306 enhancing ten times synthesis of β-galactosidase dependent on the CRP protein. Unlike P. aeruginosa, the Vfr protein in cells of P. chlororaphis 449 does not participate in the regulation of synthesis of N-acyl-homoserine lactones.     

Comparative effectiveness of <Emphasis Type="Italic">Pseudomonas</Emphasis> and <Emphasis Type="Italic">Serratia</Emphasis> sp. containing ACC-deaminase for improving growth and yield of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) under salt-stressed conditions

Ethylene synthesis is accelerated in response to various environmental stresses like salinity. Ten rhizobacterial strains isolated from wheat rhizosphere taken from different salt affected areas were screened for growth promotion of wheat under axenic conditions at 1, 5, 10 and 15 dS m⁻¹. Three strains, i.e., Pseudomonas putida (N21), Pseudomonas aeruginosa (N39) and Serratia proteamaculans (M35) showing promising performance under axenic conditions were selected for a pot trial at 1.63 (original), 5, 10 and 15 dS m⁻¹. Results showed that inoculation was effective even in the presence of higher salinity levels. P. putida was the most efficient strain compared to the other strains and significantly increased the plant height, root length, grain yield, 100-grain weight and straw yield up to 52, 60, 76, 19 and 67%, respectively, over uninoculated control at 15 dS m⁻¹. Similarly, chlorophyll content and K⁺/Na⁺ of leaves also increased by P. putida over control. It is highly likely that under salinity stress, 1-aminocyclopropane-1-carboxylic acid-deaminase activity of these microbial strains might have caused reduction in the synthesis of stress (salt)-induced inhibitory levels of ethylene. The results suggested that these strains could be employed for salinity tolerance in wheat; however, P. putida may have better prospects in stress alleviation/reduction.     

Growth characteristics and growth modeling of <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> and <Emphasis Type="Italic">Planktothrix agardhii</Emphasis> under iron limitation

Although iron is a key nutrient for algal growth just as are nitrogen and phosphorus in aquatic systems, the effects of iron on algal growth are not well understood. The growth characteristics of two species of cyanobacteria, Microcystis aeruginosa and Planktothrix agardhii, in iron-limited continuous cultures were investigated. The relationships between dissolved iron concentration, cell quota of iron, and population growth rate were determined applying two equations, Monod’s and Droop’s equations. Both species produced hydroxamate-type siderophores, but neither species produced catechol-type siderophores. The cell quota of nitrogen for both M. aeruginosa and P. agardhii decreased with decreasing cell quota of iron. The cell quota of phosphorus for M. aeruginosa decreased with decreasing cell quota of iron, whereas those for P. agardhii did not decrease. Iron uptake rate was measured in ironlimited batch cultures under different degrees of iron starvation. The results of the iron uptake experiments suggest that iron uptake rates are independent of the cell quota of iron for M. aeruginosa and highly dependent on the cell quota for P. agardhii. A kinetic model under iron limitation was developed based on the growth characteristics determined in our study, and this model predicted accurately the algal population growth and iron consumption. The model simulation suggested that M. aeruginosa is a superior competitor under iron limitation. The differences in growth characteristics between the species would be important determinants of the dominance of these algal species.     

Degradation of ethylbenzene by free and immobilized <Emphasis Type="Italic">Pseudomonas</Emphasis><Emphasis Type="Italic">fluorescens-</Emphasis>CS2

Pseudomonas fluorescens-CS2 metabolized ethylbenzene as the sole source of carbon and energy. The involvement of catechol as the hydroxylated intermediate during the biodegradation of ethylbenzene was established by TLC, HPLC and enzyme analysis. The specific activity of Catechol 2,3-dioxygenase in the cell free extracts of P. fluorescens-CS2 was determined to be 0.428 μmoles min⁻¹ mg⁻¹ protein. An aqueous-organic, Two-Phase Batch Culture System (TPBCS) was developed to overcome inhibition due to higher substrate concentrations. In TPBCS, P. fluorescens-CS2 demonstrated ethylbenzene utilization up to 50 mM without substrate inhibition on inclusion of n-decanol as the second phase. The rate of ethylbenzene metabolism in TPBCS was found enhance by fivefold in comparison with single phase system. Alternatively the alginate, agar and polyacrylamide matrix immobilized P. fluorescens-CS2 cells efficiently degraded ethylebenzene with enhanced efficiency compared to free cell cultures in single and two-phase systems. The cells entrapped in ployacrylamide and alginate were found to be stable and degradation efficient for a period of 42 days where as agar-entrapped P. fluorescens was stable and efficient a period of 36 days. This demonstrates that alginate and polyacrylamide matrices are more promising as compared to agar for cell immobilization.     

<Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> phage endolysin can enhance permeability of<Emphasis Type="Italic"> Pseudomonas aeruginosa</Emphasis> outer membrane and induce cell lysis

To determine the function of the C-terminal region of Bacillus amyloliquefaciens phage endolysin on Pseudomonas aeruginosa lysis, the permeabilization of the outer membrane of P. aeruginosa was analyzed. Glu-15 to His (E15H) and Thr-32 to Glu (T32E) substitutions were introduced into the Bacillus phage endolysin. Neither E15H nor T32E substitution induced enzymatic and antibacterial activities. These two, Glu-15 and Thr-32, were considered to be the active center of the enzyme. The addition of purified E15H and T32E proteins to P. aeruginosa cells induced the release of periplasmic -lactamase from the cells, indicating that both proteins enhance permeabilization of the outer membrane. However, the addition of E15H and T32E proteins to P. aeruginosa cells did not induce the release of cytoplasmic ATP from the cells. These results indicate that the antibacterial activity of the endolysin requires both the C-terminal enhancement of the permeabilization of the P. aeruginosa outer membrane and N-terminal enzymatic activity.     

Synthesis and biotransformation of 2-alkyl-4(1<Emphasis Type="Italic">H</Emphasis>)-quinolones by recombinant <Emphasis Type="Italic">Pseudomonas putida</Emphasis> KT2440

2-Alkyl-4(1H)-quinolones (AQs) and related derivatives, which exhibit a variety of biological properties, are secondary metabolites produced by, e.g., Pseudomonas and Burkholderia spp. Due to their main role as signaling molecules in the quorum sensing system of Pseudomonas aeruginosa, 2-heptyl-4(1H)-quinolone (HHQ) and its 3-hydroxy derivative, termed the “Pseudomonas quinolone signal” (PQS), have received considerable attention. Since chemical synthesis of different AQs is complex, we assessed the applicability of recombinant P. putida KT2440 strains for the biosynthetic production of AQs. In mineral salts medium supplemented with octanoate and anthranilate, batch cultures of P. putida KT2440 [pBBR-pqsABCD] produced about 45 μM HHQ, 30% and 70% of which were localized in the culture supernatant and methanolic cell extract, respectively. 2,4-Dihydroxyquinoline and minor amounts of C₃- to C₁₃-saturated and C_7:1 to C_13:1 monounsaturated AQs were formed as by-products. Mass spectrometry and nuclear magnetic resonance analyses spectroscopy indicated that unsaturated AQs having the same molecular mass are cis and trans isomers rather than position isomers, with the double bond located between the α and β carbon of the alkyl chain. Supplementing the cultures with hexanoate instead of octanoate shifted the AQ profile towards increased formation of C₅-AQ. Individual AQs can be prepared from concentrated methanolic extracts by preparative high-performance liquid chromatography (HPLC). Regioselective hydroxylation of HHQ to PQS can be achieved in >90% yield by biotransformation with P. putida KT2440 [pBBR-pqsH]. PQS can be isolated from methanolic cell extracts by HPLC, or be precipitated as Fe(III)-PQS complex. Preparation of a library of AQs will facilitate studies on the biological functions of these compounds.     

Pathogenicity of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> to the tobacco spider mite <Emphasis Type="Italic">Tetranychus evansi</Emphasis>

Seventeen isolates of Metarhizium anisopliae (Metschnikoff) Sorokin and two isolates of Beauveria bassiana (Balsamo) Vuillemin were evaluated for their pathogenicity against the tobacco spider mite, Tetranychus evansi Baker & Pritchard. In the laboratory all the fungal isolates were pathogenic to the adult female mites, causing mortality between 22.1 and 82.6%. Isolates causing more than 70% mortality were subjected to dose–response mortality bioassays. The lethal concentration causing 50% mortality (LC₅₀) values ranged between 0.7×10⁷ and 2.5×10⁷ conidia ml⁻¹. The lethal time to 50% mortality (LT₅₀) values of the most active isolates of B. bassiana and M. anisopliae strains varied between 4.6 and 5.8 days. Potted tomato plants were artificially infested with T. evansi and treated with B. bassiana isolate GPK and M. anisopliae isolate ICIPE78. Both fungal isolates reduced the population density of mites as compared to untreated controls. However, conidia formulated in oil outperformed the ones formulated in water. This study demonstrates the prospects of pathogenic fungi for the management of T. evansi.     

Antagonistic effect of divalent cations Ca<Superscript>2+</Superscript> and Mg<Superscript>2+</Superscript> on the morphological development of <Emphasis Type="Italic">Streptomyces hygroscopicus</Emphasis> var. <Emphasis Type="Italic">geldanus</Emphasis>

The automated docking program DOCK 5.3.0 was applied to screening for quorum sensing inhibitors (QSIs) of Peudomonus aeruginosa from a database containing 51 active components of Traditional Chinese Medicines with antibacterial activity. Five potential QSIs were revealed by the computer-based virtual screening. The compounds 3, 4, 5, 6, 7 inhibit biofilm formation of P. aeruginosa at a concentration of 200 μM. Compound 4 (baicalein) does not inhibit the growth of P. aeruginosa; however, it significantly inhibits biofilm formation of the bacteria at a lower concentration of 20 μM and promoted proteolysis of the signal receptor TraR protein in Escherichia coli at 4–40 mM. Baicalein and ampicillin showed synergistic activity against P. aeruginosa. These results suggested that baicalein can interfere with quorum sensing system of P. aeruginosa and will be developed as antibacterial agent with novel target.     

Sporulation and regeneration efficiency of phosphobacteria (<Emphasis Type="Italic">Bacillus megaterium</Emphasis> var <Emphasis Type="Italic">phosphaticum</Emphasis>)

Sporulation in Bacillus megaterium var phosphaticum (PB — 1) was induced using modified nutrient media. This modified medium induced sporulation within 36 h. After spore induction the spores were kept under refrigerated (5°C) and room temperature (32°C) for five months and survival of spores was studied at 15 days intervals by plating them in nutrient agar medium. It was observed that there was not much variation in the storage temperature (5°C & 32°C). The spore cells of Bacillus megaterium var phosphaticum (PB — 1) were observed up to five months of storage under refrigerated (5°C) and room temperature (32°C). Regeneration of spore cells into vegetative cells was studied in tap water, rice gruel, nutrient broth, sterile lignite and sterile water at different concentrations of spore inoculum. The multiplication of sporulated Bacillus megaterium var phosphaticum culture was fast and reached its maximum (29.5 × 10⁸ cfu ml⁻¹) in nutrient broth containing 5 per cent inoculum level.