Distribution of <Emphasis Type="Italic">Nevskia ramosa</Emphasis> and Other Rosette-Forming Neustonic Bacteria

Samples from 27 natural and artificial aquatic environments were analyzed for the presence of rosette-forming bacteria by a combined cultivation and molecular biological approach. Rosette-forming bacteria developed in 20 enrichment cultures with ammonia-free medium under air. Three morphotypes could be distinguished. The most abundant type I resembled Nevskia ramosa and formed hydrophobic, flat, and dichotomously branching rosettes. Type II rosettes were three-dimensional and were observed in 10 enrichments, often together with those of type I. These rosettes were hydrophilic indicating life in the hyponeuston underneath the air–water interface. Rosettes of a third type consisted of hydrophilic slime stalks that were excreted at the cell poles and were observed in only one sample. Using fluorescence in situ hybridization (FISH) with the Nevskia-specific probes NEV177 and NEV656, the presence of Nevskia ramosa was demonstrated in exactly those samples that showed type I rosettes. In a series of most-probable-number experiments, during a calm and sunny weather period 430,000 Nevskia-like bacteria per mL were found in surface samples, while during rainy weather and within the water body the numbers were lower by several orders of magnitude. Five pure cultures isolated from various enrichments were characterized in detail. The two isolates forming type I rosettes were identified as Nevskia ramosa by 16S rDNA analysis. However, comparison by genomic fingerprinting (ERIC-PCR) revealed differences between the two isolates and previously characterized strains. The 16S rDNA of two isolates forming type II rosettes showed 97.6% similarity to that of Pseudomonas fluorescens. The closest relative of the isolate forming type III rosettes was Sphingomonas parapaucimobilis (96.4% sequence similarity of the 16S rRNA sequence). All isolates grew homogeneously submersed if ammonia was added to the medium. Our results indicate that Nevskia ramosa is a widely distributed epineustonic bacterium, which can specifically be deleted by its flat and hydrophobic rosettes on ammonia-free media.     

Effects of Ultraviolet Radiation on the Gram-Positive Marine Bacterium <Emphasis Type="Italic">Microbacterium maritypicum</Emphasis>

Although extensive information is available on the effect ultraviolet (UV) radiation has on Gram-negative marine bacteria, there is a scarcity of data concerning UV radiation and Gram-positive marine bacteria. The focus of this paper is on Microbacterium maritypicum, with the Gram-negative Vibrio natriegens being used as a standard of comparison. M. maritypicum exhibited growth over a NaCl range of 0–1000 mM, with optimum growth occurring between 0 and 400 mM NaCl. In contrast, V. natriegens grew over a NaCl span of 250–1000 mM, with best growth being observed between 250 and 600 mM NaCl. UV radiation experiments were done using the medium with 250 mM NaCl. For solar (UV-A and B) radiation and log-phase cells, M. maritypicum was determined to be three times more resistant than V. natriegens. For germicidal (UV-C) radiation, the pattern of resistance of the log-phase cells to the lethal effects of the radiation was even more pronounced, with the Gram-positive bacterium being more than 12 to 13 times more resistant. Similar data to the solar and germicidal log-phase UV kill curves were obtained for stationary-phase cells of both organisms. Photoreactivation was observed for both types of cells exposed to UV-C but none for cells treated with UV-A and B. When log phase cells of M.maritypicum were grown at 0.0 and 0.6 M NaCl and exposed to UV-C radiation, no difference in survivorship patterns was noted from that of 0.25 M NaCl grown cells. Although this study has only focused on two marine bacteria, our results indicate that the Gram-positive M. maritypicum could have a built-in advantage for survival in some marine ecosystems.     

Composition,secretion kinetics and radioactive labelling of the extracellular slime polysaccharide secreted during spherulation ofPhysarum polycephalum

The myxomycetePhysarum polycephalum synthesizes copious amounts of slime when differentiating into the hard walled resting stage. The chemical composition of slime obtained after introduction of spherulation in a nutrient and a non-nutrient salt-medium has been analysed and compared. The composition of slime is almost identical after the two different induction methods. This slime could be labelled with radioactived-[U-¹⁴C]glucose,³²PO₄ ^3–,³⁵SO₄ ^2– and⁷⁵SeO₄ ^2–. The kinetics of slime secretion after both induction methods has been followed using different criteria. The sulfate analog⁷⁵SeO₄ ^2– seems to be incorporated into the slime, partially replacing sulfate groups of the sulfated polysaccharide. Furthermore,d-[U-¹⁴C]glucose was used to labe the spherule walls. Determination of an alkali resistent polysaccharide component serves as a new method to follow wall formation.     

<Emphasis Type="SmallCaps">d</Emphasis>-amino acids for the enhancement of a binary biocide cocktail consisting of THPS and EDDS against an SRB biofilm

Biofilms of sulfate reducing bacteria (SRB) are often responsible for Microbiologically Influenced Corrosion (MIC) that is a major problem in the oil and gas industry as well as water utilities and other industries. This work was inspired by recent reports that some d-amino acids may be useful in the control of microbial biofilms. A d-amino acid mixture with equimolar d-tyrosine, d-methionine, d-tryptophan and d-leucine was tested in this work for their enhancement of a biocide cocktail containing tetrakis (hydroxymethyl) phosphonium sulfate (THPS) and ethylenediamine-N,N’-disuccinic acid (EDDS). Desulfovibrio vulgaris (ATCC 7757) was cultured in ATCC 1249 medium. Its biofilm was grown on C1018 carbon steel coupons. Experimental results indicated that the triple biocide cocktail consisting of 30 ppm THPS, 500 ppm EDDS and 6.6 ppm d-amino acid mixture (with equimolar d-tyrosine, d-methionine, d-tryptophan and d-leucine) was far more effective than THPS and EDDS alone and their binary combination. The triple biocide cocktail effectively prevented SRB biofilm establishment and removed the established SRB biofilm. The d-amino acid mixture alone did not show significant effects in the two tasks even at 660 ppm.     

Comparative Genomic and Phylogenetic Analyses Reveal the Evolution of the Core Two-Component Signal Transduction Systems in Enterobacteria

The two-component signal transduction system (TCST) consists of a histidine kinase (HK) and a response regulator (RR). TCSTs play important roles in sensing and reacting to environmental changes, and in bacterial pathogenesis. Previously, we have identified and characterized TCSTs in Erwinia amylovora, a severe plant enterobacterial pathogen, at genome-wide level. Here we conducted a comparative genomic analysis of TCSTs in 53 genomes of 16 enterobacterial species. These species include important plant, animal, human, and insect pathogenic, saprophytic or symbiotic microorganisms. Comparative genomic analysis revealed that enterobacteria contain eight pairs of core TCSTs. Phylogenetic trees reconstructed from a concatenation of the core set of TCSTs from enterobacteria and for individual TCST proteins from species in Proteobacteria showed that most TCST protein trees in the Enterobacteriaceae or in species of the γ-Proteobacteria agreed well with that of the corresponding 16S rRNA gene. It also showed that co-evolutionary relationships existed between cognate partners of the HKs and RRs. Several core TCSTs were quite ancient and universal based on phylogenomic analysis of protein structures. These results indicate that the core TCSTs are relatively conserved, and suggest that these enterobacteria may have maintained their ancient core TCSTs and might acquire specific new TCSTs for their survival in different environments or hosts, or may have evolved new functionalities of the core TCSTs for adaptation to different ecological niches.     

Endophytic and rhizospheric enterobacteria isolated from sugar cane have different potentials for producing plant growth-promoting substances

Background and aims

Endophytic and rhizospheric environments differ in many respects, leading to the presence of different bacterial communities at each site. However, microorganisms such as enterobacteria can be found both within plants and in the surrounding soil. Bacteria must present differences in the traits that affect such environments in order to successfully colonise them. The present study compared the plant growth-promoting potential of diazotrophic enterobacteria isolated from the rhizosphere and from within surface-disinfected plants.

Methods

A total of 46 diazotrophic enterobacterial strains (21 rhizospheric and 25 putatively endophytic) belonging to the Klebsiella and Enterobacter genera, which are prevalent in sugar cane plantations, were isolated from the rhizosphere and from surface-disinfected plants. Their ability to synthesise amino acids using combined nitrogen obtained from nitrogen fixation, and their ability to synthesise indole-3-acetic acid (IAA) were determined by high performance liquid chromatography. Endogenous ethylene production by the bacteria was measured using gas chromatography, and biocontrol of phytopathogenic fungi was determined qualitatively using a dual culture technique.

Results

The putative endophytes released significantly higher amounts of amino acids than the rhizospheric bacteria, whilst the latter produced higher quantities of ethylene and were more actively antagonistic to fungi. Both types of bacteria released similar amounts of IAA.

Conclusion

Endophytic and rhizospheric bacteria differ in their capacity to release plant growth-promoting substances, which may be a reflection of their adaptations and an indication of their potential impact on their natural environment.

     

Cultural and biochemical diversity of pink-pigmented bacteria isolated from paper mill slimes

A study of 25 paper mill slime deposits and one additive revealed nine pink-pigmented bacterial isolates, eight of which were different from pink-pigmented bacteria identified in the paper industry in the middle 1900s. The pink-pigmented bacteria described previously in pulp and paper included Micrococcus agilis, Bacillus subtilis, Serratia sp. and Alcaligenes viscosus. With the exception of one isolate, Micrococcus sp., these isolates possessed many cultural, biochemical and chemical properties which were different from the ones previously reported for paper mills. Eight of these bacteria were Gram-negative rods or filamentous, aerobic and positive for catalase production. Two isolates were methylotrophic, oxidizing methanol and identified as Methylobacterium zatmanii. Cellular fatty acid analysis and other characteristics showed one isolate to be Roseomonas sp. Using 16S rRNA gene sequencing, one isolate which was a Gram-negative rod was identified as Deionococcus grandis. Four bacteria had cells that were long or filamentous and these were isolated from mills with pink slime problems. The identity of one of the filamentous bacteria was determined by 16S rRNA gene sequencing to be close to Flectobacillus sp. strain MWH38. Most of the isolates were susceptible to 11 industrial biocides. Journal of Industrial Microbiology & Biotechnology (2000) 25, 74–80. Received 28 January 2000/ Accepted in revised form 09 June 2000     

<Emphasis Type="SmallCaps">d</Emphasis>-Hexosaminate production by oxidative fermentation

Microbial production of d-hexosaminate was examined by means of oxidative fermentation with acetic acid bacteria. In most strains of acetic acid bacteria, membrane-bound d-glucosamine dehydrogenase (synonymous with an alternative d-glucose dehydrogenase distinct from quinoprotein d-glucose dehydrogenase) oxidized d-hexosamines to the corresponding d-hexosaminates in a stoichiometric manner. Conversion of d-hexosamines to the corresponding d-hexosaminates was observed with growing cells of acetic acid bacteria, and d-hexosaminate was stably accumulated in the culture medium even though d-hexosamine was exhausted. Since the enzyme responsible is located on the outer surface of the cytoplasmic membrane, and the enzyme activity is linked to the respiratory chain of the organisms, resting cells, dried cells, and immobilized cells of acetic acid bacteria were effective catalysts for d-hexosaminate production. d-Mannosaminate and d-galactosaminate were also prepared for the first time by means of oxidative fermentation, and three different d-hexosaminates were isolated from unreacted substrate by a chromatographic separation. In this paper, d-hexosaminate production by oxidative fermentation carried out mainly with Gluconobacter frateurii IFO 3264 is exemplified as a typical example.     

Identification of bacteria contaminating pulp and a paper machine in a Canadian paper mill

Over 100 bacteria from pulp and slime samples in a Canadian paper mill were identified by partial sequencing of their 16S rDNAs. Seventy-one percent of the isolates could be assigned to a bacterial genus with a high level of confidence. Another 12% exhibited at least 95% similarity within their 16S rDNA sequence with unidentified organisms that originate from warm or wet environments. Pseudomonas, Bacillus, and Pseudoxanthomonas isolates were represented at a relatively high proportion in both pulp and slime samples. This is the first time that Pseudoxanthomonas strains have been isolated from pulp and slime samples on a paper machine. Electronic Publication     

Nutritional studies on species and mutants of Lepista,Cantharellus, Pleurotus and Volvariella

The purpose of this study is to determine the ability of select Agaricales species to utilize various sources of carbon, nitrogen, vitamins, and growth hormones. Fungi selected for the studies include:Cantharellus clavatus Fries,C. cibarius Fries,Lepista nuda (Bull. ex. Fries)Cooke,Pleurotus ostreatus (Jacq. ex. Fries)Kummer, andVolvariella volvacea (Bull. ex. Fries)Singer. Three strains ofC. cibarius and one mutant ofV. volvacea (V135), V134, were employed to determine if nutritional requirement differences occurred. One species,V. volvacea, is grown commercially as a cottage industry in the Orient (Alicbusan &Ela, 1961) while the other species currently have no commercial value. All species studied possess pleasing flavors and have potential use in the mushroom production industry.A literature compilation of the nutritional regulation of basidiocarp formation and vegetative growth of Agaricales was made with specific mention to the named species (Volz &Beneke, 1969). Recent nutritional studies with one or more of the specific species include those byYusef &Allam (1967), andEger (1970).     

Comparison of the biological activity of fusicoccin in higher plants with its binding to plasma membranes

The concentration dependences of the binding of fusicoccins (FCs) A, B, C, D, J and H to plasma membranes isolated from maize (Zea mays L.) roots have been studied in parallel with the effects of these compounds on elongation and ⁸⁶Rb transport in detached maize roots. The dissociation constants obtained showed a good correlation between the affinity of the FCs for the plasmalemma and their biological activity. However, the range of physiologically active FC concentrations proved to be about two orders of magnitude higher than that calculated from the dissociation constants. It was also shown that Vicia faba L. mesophyll protoplasts, unlike isolated plasma membranes, have two FC-binding sites, one with a K _D similar to that of the isolated plasmalemma while the other has a substantially higher K _D , apparently corresponding to the physiologically active state of the FC-binding proteins.Abbreviation FC fusicoccin     

Influence of a Probiotic Enterococcus Faecium Strain on Selected Bacterial Groups in the Small Intestine of Growing Turkey Poults

A feeding trial was carried out with turkey poults, which were fed a diet containing 10¹⁰ viable probiotic E. faecium NCIB 10415 cells/kg feed. Samples of the intestinal tract were analyzed for lactate, colony forming units of total anaerobic bacteria, lactic acid bacteria, enterobacteria and enterococci. Furthermore, metabolic activity of total eubacterial, lactobacilli and enterococci was recorded in selected RNA-extracts with specific ribosomal RNA oligonucleotide probes. Animals fed the probiotic diet showed continously increasing lactate concentrations throughout the sampling period up to day 42 of life. No correlation was found for colony forming units (cfu) of lactic acid bacteria, but metabolic activity of lactobacilli showed very close relation to continously increasing lactate concentrations. Throughout the feeding trial, enterococci in the control group continously increased to a maximum of 10⁴ cfu/g wet weight, but 10-fold higher enterococci cfu were generally found in the treated group. However, rRNA content as measure for metabolic activity showed a drastic decline in both groups after high metabolic activities on day 7. This study shows that E. faecium NCIB 10415 (E. faecium SF68) stimulates other lactic acid bacteria in the small intestine, especially lactobacilli.     

Characterization of new <Emphasis Type="SmallCaps">l,d</Emphasis>-endopeptidase gene product CwlK (previous YcdD) that hydrolyzes peptidoglycan in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Bacillus subtilis has various cell wall hydrolases, however, the functions and hydrolase activities of some enzymes are still unknown. B. subtilis CwlK (YcdD) exhibits high sequence similarity with the peptidoglycan hydrolytic l,d-endopeptidase (PLY500) of Listeria monocytogenes phage and CwlK has the VanY motif which is a d-alanyl-d-alanine carboxypeptidase (Pfam: http://www.sanger.ac.uk/Software/Pfam/). The β-galactosidase activity observed on cwlK-lacZ fusion indicated that the cwlK gene was expressed during the vegetative growth phase, and Western blotting suggested that CwlK seems to be localized in the membrane. Truncated CwlK fused with a histidine-tag (h-ΔCwlK) was produced in Escherichia coli and purified on a nickel column. The h-ΔCwlK protein hydrolyzed the peptidoglycan of B. subtilis, and the optimal pH, temperature and NaCl concentration for h-ΔCwlK were pH 6.5, 37°C, and 0 M, respectively. Interestingly, h-ΔCwlK could hydrolyze the linkage of l-alanine-d-glutamic acid in the stem of the peptidoglycan, however, this enzyme could not hydrolyze the linkage of d-alanine-d-alanine, suggesting that CwlK is an l,d-endopeptidase not a d,d-carboxypeptidase. CwlK could not hydrolyze polyglutamate from B. natto or peptidoglycan of Staphylococcus aureus. This is the first report describing the characterization of an l,d-endopeptidase in B. subtilis and also the first report in bacteria of the characterization of a PLY500 family protein encoded in chromosomal DNA. Tatsuya Fukushima and Yang Yao contributed equally to this work.     

Bacterial community structure and colonization patterns of Fagus sylvatica L. ectomycorrhizospheres as determined by fluorescence in situ hybridization and confocal laser scanning microscopy

) grown in natural forest soil in southern Germany was examined by fluorescence in situ hybridization (FISH) using fluorescent oligonucleotide probes, targeting phylogenetic relevant sequences of the 16S and 23S rRNA. Lactarius subdulcis, L. vellereus, L. rubrocinctus and Laccaria amethystina were found to be the prevalent fungi forming ectomycorrhizae with F. sylvatica. For FISH studies using confocal laser scanning microscopy, oligonucleotide probes labeled with carboxymethylindocyanine-succinimidyl ester allowed detection of associated bacteria, because the autofluorescence of ectomycorrhiza samples could be overcome in the infrared. Bacteria of the α-, β and γ-subclasses of the proteobacteriawere detected in high numbers on mantle surfaces, while members of other phylogenetically defined groups were found in smaller numbers. This contrasts with previous published results on the cultivation of mycorrhiza-associated bacteria. Hybridizing bacteria were also found within damaged cells of the hyphal mantle of L. rubrocinctus, as well as on emanating hyphae of L. amethystina. Using a newly developed extraction protocol for bacteria associated with ectomycorhizas, the two most common fungi on F. sylvatica, L. vellereus and L. subdulcis, were mostly associated with members of the α- and β-subclasses of the proteobacteria. The proportion of hybridizing bacteria varied between the two ectomycorrhizae, which were thus host to distinct populations of bacteria.     

Cyanide formation in preparations from Chlorella and New Zealand spinach leaves: Effect of added amino acids

Summary As part of an effort to identify the natural precursor(s) of HCN in the alga Chlorella vulgaris Beijerinck, and in leaves of New Zealand spinach (Tetragonia expansa, Murr.), HCN release was measured after addition of various amino acids to illuminated algal extracts and grana preparations. Histidine is particularly effective as an HCN precursor, both with Chlorella extracts and leaf grana. With the algal extracts, d-histidine is about ten times more effective than l-histidine and histamine, whereas the two isomers (and histamine) are about equally effective with leaf grana. In the presence of leaf grana plus added Mn²⁺ and peroxidase, l-tyrosine and l-cysteine like-wise cause HCN formation; but these amino acids cause little or no HCN formation in the presence of Chlorella extracts. A stimulation of HCN production by l-histidine was observed with intact Chlorella cells. Because of the limitations of the assay method, the possibility can not be excluded that other substances than histidine may also lead to HCN generation in Chlorella vulgaris, but the results show that histidine has an important role in HCN generation by this species.Abbreviation POD peroxidase     

Xylose metabolism in the anaerobic fungus<Emphasis Type="Italic"> Piromyces</Emphasis> sp. strain E2 follows the bacterial pathway

The anaerobic fungus Piromyces sp. strain E2 metabolizes xylose via xylose isomerase and d-xylulokinase as was shown by enzymatic and molecular analyses. This resembles the situation in bacteria. The clones encoding the two enzymes were obtained from a cDNA library. The xylose isomerase gene sequence is the first gene of this type reported for a fungus. Northern blot analysis revealed a correlation between mRNA and enzyme activity levels on different growth substrates. Furthermore, the molecular mass calculated from the gene sequence was confirmed by gel permeation chromatography of crude extracts followed by activity measurements. Deduced amino acid sequences of both genes were used for phylogenetic analysis. The xylose isomerases can be divided into two distinct clusters. The Piromyces sp. strain E2 enzyme falls into the cluster comprising plant enzymes and enzymes from bacteria with a low G+C content in their DNA. The d-xylulokinase of Piromyces sp. strain E2 clusters with the bacterial d-xylulokinases. The xylose isomerase gene was expressed in the yeast Saccharomyces cerevisiae, resulting in a low activity (25±13 nmol min^–1mg protein^-1). These two fungal genes may be applicable to metabolic engineering of Saccharomyces cerevisiae for the alcoholic fermentation of hemicellulosic materials.     

Achene morphology and slime structure in some taxa of Artemisia L. and Neopallasia L. (Asteraceae)

We have examined slime cell distribution on the surface of the achenes of some Artemisia and Neopallasia taxa, as well as slime composition, envelope formation during the hydration, and slime relation to different morphological features and environmental factors. The results of the studies show a characteristic pattern of slime cells distribution, which could differ between taxa. The slime in the taxa studied belongs to the cellulose type and consists of two components i.e., pectins and cellulose. Although all fruits contain slime cells, not all of them show the slime envelope formation. Plants occurring in dry habitats (such as A. barrelieri) or annual species (such as A. annua) are characterised by a large amount of slime and a fast process of slime envelope formation. Slime production has not been observed in some polyploid populations (A. campestris and A. campestris ssp. sericea) and in two species occurring in relatively fertile habitats (A. verlotiorum, A. vulgaris). A reason for this may be either the immaturity of polyploid fruits leading to the production of a scarce, not detectable slime amount or, alternatively, the occurrence of not functional slime cells. Slime facilitates and stimulates the germination, as well as the adherence of the fruits to the ground or to animals (for dispersal). The slime could play important role in the distribution and colonisation of new habitats in many Artemisia taxa.     

Ultrastructure of the plasmodial slime moldPerichaena vermicularis

Summary The development of the peridium ofPerichaena vermicularis has been examined using light and electron microscopy and acid phosphatase localization. A newly formed fruiting body consists of undifferentiated protoplasm which is enveloped by a slime coat. Almost immediately after formation of the plasmodiocarp, the protoplasm differentiates into autolytic and fruiting regions. The autolytic region is located at irregular intervals between the slime coat and the fruiting region and separated from both of them by membranes.Soon after the autolytic region has formed, additional signs of degeneration appear in the autolytic region including unusual appearance of nuclei, increase in autophagic vacuoles, and the presence of clear areas in the ground substance. The plasma membrane, which once completely separated the slime coat from the autolytic region, is no longer continuous. Electron micrographs of the autolytic region from later developmental stages show formation of extensive channels which contain protoplasm in various stages of degradation. Acid phosphatase is present in the channels of the autolytic region. The morphological evidence and the presence of hydrolytic enzyme suggest the region is being digested and re-adsorbed.After the autolytic region has been digested, an even layer of peridial wall material is laid down, and at regular intervals additional wall material is produced. The additional wall material forms the reticulation on the inside of the peridial wall.This work was supported by National Sciences Foundation grants (GB-5883 and GB-8537) to Dr.Ian K.Ross and an NSF Traineeship (GZ 445 and 796) to I.Charvat.This constitutes a portion of a thesis presented to the Regents of the University of California by the first author in partial fulfillment of the requirements for the Ph. D. degree.     

Diketopiperazines Produced by the Halophilic Archaeon, <Emphasis Type="Italic">Haloterrigena hispanica</Emphasis>, Activate AHL Bioreporters

The generic term “quorum sensing” has been adopted to describe the bacterial cell-to-cell communication mechanism which coordinates gene expression when the population has reached a high cell density. Quorum sensing depends on the synthesis of small molecules that diffuse in and out of bacterial cells. There are few reports about this mechanism in Archaea. We report the isolation and chemical characterization of small molecules belonging to class of diketopiperazines (DKPs) in Haloterrigena hispanica, an extremely halophilic archaeon. One of the DKPs isolated, the compound cyclo-(l-prolyl–l-valine) activated N-acyl homoserine lactone (AHL) bioreporters, indicating that Archaea may have the ability to interact with AHL-producing bacteria within mixed communities.