Effects of salinity on antibiotic production in sponge-derived Salinispora actinobacteria.

AIMS: To investigate the effects of growth conditions related to marine habitat on antibiotic production in sponge-derived Salinispora actinobacteria. METHODS AND RESULTS: Media with varying salt concentration were used to investigate the effects of salinity in relation to Salinispora growth and rifamycin production. The chemotypic profiles of the model strain Salinispora arenicola M413 was then assessed using metabolomic fingerprints from high-pressure liquid chromatography with diode array detection (HPLC-DAD) and multivariate data analysis, before extending this approach to two other strains of S. arenicola. Fingerprint data were generated from extracts of S. arenicola broth cultures grown in media of varying salt (NaCl) concentrations. These fingerprints were then compared using multivariate analysis methods such as principal components analysis (PCA) and orthogonal projection to latent structures discriminant analysis (OPLS-DA). From the analysis, a low-sodium growth condition (1% NaCl) was found to delay the onset of growth of the model S. arenicola M413 strain when compared to growth in media with either 3% artificial sea salt or 3% NaCl. However, low-sodium growth conditions also increased cell mass yield and contributed to at least a significant twofold increase in rifamycin yield when compared to growth in 3% artificial sea salt and 3% NaCl. CONCLUSIONS: The integration of HPLC-DAD and multivariate analysis proved to be an effective method of assessing chemotypic variations in Salinispora grown in different salt conditions, with clear differences between strain-related chemotypes apparent due to varying salt concentrations. SIGNIFICANCE AND IMPACT OF THE STUDY: The observed variation in S. arenicola chemotypic profiles further suggests diversity in secondary metabolites in this actinomycete in response to changes in the salinity of its environment.

Diversity and distribution of the bioactive actinobacterial genus Salinispora from sponges along the Great Barrier Reef.

Isolates from the marine actinobacterial genus Salinispora were cultured from marine sponges collected from along the length of the Great Barrier Reef (GBR), Queensland, Australia. Strains of two species of Salinispora, Salinispora arenicola and "Salinispora pacifica", were isolated from GBR sponges Dercitus xanthus, Cinachyrella australiensis and Hyattella intestinalis. Phylogenetic analysis of the 16S rRNA gene sequences of representative strains, selected via BOX-PCR screening, identified previously unreported phylotypes of the species "S. pacifica". The classification of these microdiverse 16S rRNA groups was further confirmed by analysis of the ribonuclease P RNA (RNase P RNA) gene through both phylogenetic and secondary structure analysis. The use of RNase P RNA sequences combined with 16S rRNA sequences allowed distinction of six new intraspecies phylotypes of "S. pacifica" within the geographical area of the GBR alone. One of these new phylotypes possessed a localised regional distribution within the GBR.

Anaerobic ammonium oxidation by marine and freshwater planctomycete-like bacteria.

Recently, two fresh water species, " Candidatus Brocadia anammoxidans" and " Candidatus Kuenenia stuttgartiensis", and one marine species, " Candidatus Scalindua sorokinii", of planctomycete anammox bacteria have been identified. " Candidatus Scalindua sorokinii" was discovered in the Black Sea, and contributed substantially to the loss of fixed nitrogen. All three species contain a unique organelle--the anammoxosome--in their cytoplasm. The anammoxosome contains the hydrazine/hydroxylamine oxidoreductase enzyme, and is thus the site of anammox catabolism. The anammoxosome is surrounded by a very dense membrane composed almost exclusively of linearly concatenated cyclobutane-containing lipids. These so-called 'ladderanes' are connected to the glycerol moiety via both ester and ether bonds. In natural and man-made ecosystems, anammox bacteria can cooperate with aerobic ammonium-oxidising bacteria, which protect them from harmful oxygen, and provide the necessary nitrite. The cooperation of these two groups of ammonium-oxidising bacteria is the microbial basis for a sustainable one reactor system, CANON (completely autotrophic nitrogen-removal over nitrite) to remove ammonia from high strength wastewater.

Cell compartmentalisation in planctomycetes: novel types of structural organisation for the bacterial cell.

The organisation of cells of the planctomycete species Pirellula marina, Isosphaera pallida, Gemmata obscuriglobus, Planctomyces maris and "Candidatus Brocadia anammoxidans" was investigated based on ultrastructure derived from thin-sections of cryosubstituted cells, freeze-fracture replicas, and in the case of Gemmata obscuriglobus and Pirellula marina, computer-aided 3-D reconstructions from serial sections of cryosubstituted cells. All planctomycete cells display a peripheral ribosome-free region, termed here the paryphoplasm, surrounding the perimeter of the cell, and an interior region including any nucleoid regions as well as ribosome-like particles, bounded by a single intracytoplasmic membrane (ICM), and termed the pirellulosome in Pirellula species. Immunogold labelling and RNase-gold cytochemistry indicates that in planctomycetes all the cell DNA is contained wholly within the interior region bounded by the ICM, and the paryphoplasm contains no DNA but at least some of the cell's RNA. The ICM in Isosphaera pallida and Planctomyces maris is invaginated such that the paryphoplasm forms a major portion of the cell interior in sections, but in other planctomycetes it remains as a peripheral zone. In the anaerobic ammonium-oxidising ("anammox" process) chemoautotroph "Candidatus Brocadia anammoxidans" the interior region bounded by ICM contains a further internal single-membrane-bounded region, the anammoxosome. In Gemmata obscuriglobus, the interior ICM-bounded region contains the nuclear body, a double-membrane-bounded region containing the cell's nucleoid and all genomic DNA in addition to some RNA. Shared features of cell compartmentalisation in different planctomycetes are consistent with the monophyletic nature of the planctomycetes as a distinct division of the Bacteria. The shared organisational plan for the planctomycete cell constitutes a new type not known in cells of other bacteria.

Phylogenetic analysis of evolutionary relationships of the planctomycete division of the domain bacteria based on amino acid sequences of elongation factor Tu.

Sequences from the tuf gene coding for the elongation factor EF-Tu were amplified and sequenced from the genomic DNA of Pirellula marina and Isosphaera pallida, two species of bacteria within the order Planctomycetales. A near-complete (1140-bp) sequence was obtained from Pi. marina and a partial (759-bp) sequence was obtained for I. pallida. Alignment of the deduced Pi. marina EF-Tu amino acid sequence against reference sequences demonstrated the presence of a unique 11-amino acid sequence motif not present in any other division of the domain Bacteria. Pi. marina shared the highest percentage amino acid sequence identity with I. pallida but showed only a low percentage identity with other members of the domain Bacteria. This is consistent with the concept of the planctomycetes as a unique division of the Bacteria. Neither primary sequence comparison of EF-Tu nor phylogenetic analysis supports any close relationship between planctomycetes and the chlamydiae, which has previously been postulated on the basis of 16S rRNA. Phylogenetic analysis of aligned EF-Tu amino acid sequences performed using distance, maximum-parsimony, and maximum-likelihood approaches yielded contradictory results with respect to the position of planctomycetes relative to other bacteria. It is hypothesized that long-branch attraction effects due to unequal evolutionary rates and mutational saturation effects may account for some of the contradictions.

Microbiology and application of the anaerobic ammonium oxidation ('anammox') process.

Ten years ago, an anaerobic ammonium oxidation ('anammox') process was discovered in a denitrifying pilot plant reactor. From this system, a highly enriched microbial community was obtained, dominated by a single deep-branching planctomycete, Candidatus Brocadia anammoxidans. Phylogenetic inventories of different wastewater treatment plants with anammox activity have suggested that at least two genera in Planctomycetales can catalyse the anammox process. Electron microscopy of the ultrastructure of B. anammoxidans has shown that several membrane-bounded compartments are present inside the cytoplasm. Hydroxylamine oxidoreductase, a key anammox enzyme, is found exclusively inside one of these compartments, tentatively named the 'anammoxosome'.

Na(+)/H(+)exchangers: linking osmotic dysequilibrium to modified cell function.

The Na(+)/H(+) exchangers (NHEs) are among the major ion transporters involved in cell volume regulation. NHE activation leads to a cellular influx of Na(+) ions and extrusion of H(+) ions, which are readily replenished from intracellular buffers. This will result in a net import of Na(+). In many systems NHE operates in parallel to Cl(-)/ HCO3(-) exchange, resulting in cellular uptake of NaCl. The influx of osmotically obliged water will consequently lead to cell swelling. This makes NHEs suitable to serve as powerful mechanisms for increasing cell volume (CV). The low volume threshold for NHE activation enables the cells to respond to very minute reductions of the CV. By the coupling to the export of H(+) ions cell volume regulatory NHE activation may lead to changes in intracellular pH. On the other hand NHEs are activated by a broad variety of ligands and by intracellular acidosis, which, in turn, may consequently lead to cell swelling. In addition, NHEs are linked to other intracellular proteins and structures, like e.g. the cytoskeleton, which themelves are involved in the regulation of numerous cellular processes. Therefore NHEs link CV regulation to a diversity of cellular functions, both in physiological and pathophysiological conditions. Six isoforms of the Na(+)/H(+) exchanger, termed NHE1--6, have been cloned so far. NHE 1--5 are located in the plasma membrane, whereas NHE6 is sorted to the mitochondrial membrane. NHE1 and NHE6 are the ubiquitously expressed isoforms. The expression of the isoforms NHE2 to NHE5 is restricted to specific tissues and the pattern of their expression, as well as their subcellular localization indicate that they fulfill specialized functions. Cell shrinkage induced activation has been shown for NHE1,2 and 4. In contrast, NHE3 is inhibited by cell shrinkage. In many cells several isoforms are present and assigned to specific membrane domains where they may serve a functional crosstalk between the different ion transporters.

Missing lithotroph identified as new planctomycete.

With the increased use of chemical fertilizers in agriculture, many densely populated countries face environmental problems associated with high ammonia emissions. The process of anaerobic ammonia oxidation ('anammox') is one of the most innovative technological advances in the removal of ammonia nitrogen from waste water. This new process combines ammonia and nitrite directly into dinitrogen gas. Until now, bacteria capable of anaerobically oxidizing ammonia had never been found and were known as "lithotrophs missing from nature". Here we report the discovery of this missing lithotroph and its identification as a new, autotrophic member of the order Planctomycetales, one of the major distinct divisions of the Bacteria. The new planctomycete grows extremely slowly, dividing only once every two weeks. At present, it cannot be cultivated by conventional microbiological techniques. The identification of this bacterium as the one responsible for anaerobic oxidation of ammonia makes an important contribution to the problem of unculturability.

Effect of active immunization against luteinizing hormone-releasing hormone on the androgen-sensitive Dunning R3327-PAP and androgen-independent Dunning R3327-AT2.1 prostate cancer sublines.

BACKGROUND: The objective of this study was to determine the effect of active immunization against LHRH on the growth characteristics and histology of subcutaneously implanted tumors of the androgen-sensitive Dunning R3327-PAP and androgen-independent R3327-AT2.1 rat prostate adenocarcinoma sublines. RESULTS: We herein demonstrate that 1) active immunization with an LHRH-diphtheria toxoid-conjugate (LHRH-DT) leads to the downregulation of gonadotropins and testosterone and consequently the atrophy of testosterone-dependent organs such as the testes, prostate, and androgen-sensitive Dunning R3327-PAP tumors, 2) growth inhibition of Dunning R3327-PAP tumors is caused by suppression of cell division rather than by an increase in cell death and is associated with an increase of the tumor stroma content, and 3) volume increase of the androgen-independent Dunning R3327-AT2.1 tumor is slightly but significantly reduced, indicating a local stimulatory LHRH loop within this tumor cell line.

Isolation and molecular identification of planctomycete bacteria from postlarvae of the giant tiger prawn, Penaeus monodon.

Bacteria phenotypically resembling members of the phylogenetically distinct planctomycete group of the domain Bacteria were isolated from postlarvae of the giant tiger prawn, Penaeus monodon. A selective medium designed in the light of planctomycete antibiotic resistance characteristics was used for this isolation. Planctomycetes were isolated from both healthy and monodon baculovirus-infected prawn postlarvae. The predominant colony type recovered from postlarvae regardless of viral infection status was nonpigmented. Other, less commonly observed types were pink or orange pigmented. A planctomycete-specific 16S rRNA-directed probe was designed and used to screen the isolates for their identity as planctomycetes prior to molecular phylogenetic characterization. 16S rRNA genes from nine prawn isolates together with two planctomycete reference strains (Planctomyces brasiliensis and Gemmata obscuriglobus) were sequenced and compared with reference sequences from the planctomycetes and other members of the domain Bacteria. Phylogenetic analyses and sequence signatures of the 16S rRNA genes demonstrated that the prawn isolates were members of the planctomycete group. Five representatives of the predominant nonpigmented colony type were members of the Pirellula group within the planctomycetes, as were three pink-pigmented colony type representatives. Homology values and tree topology indicated that representatives of the nonpigmented and pink-pigmented colony types formed two discrete clusters within the Pirellula group, not identical to any known Pirellula species. A sole representative of the orange colony type was a member of the Planctomyces group, virtually identical in 16S rDNA sequence to P. brasiliensis, and exhibited distinctive morphology.

Phylogenetic positions of novel aerobic, bacteriochlorophyll a-containing bacteria and description of Roseococcus thiosulfatophilus gen. nov., sp. nov., Erythromicrobium ramosum gen. nov., sp. nov., and Erythrobacter litoralis sp. nov.

We analyzed the 16S ribosomal DNAs of three obligately aerobic, bacteriochlorophyll a-containing bacteria, "Roseococcus thiosulfatophilus," "Erythromicrobium ramosum," and new isolate T4T (T = type strain), which was obtained from a marine cyanobacterial mat. "Roseococcus thiosulfatophilus" is a member of the alpha-1 subclass of the Proteobacteria and is moderately related to Rhodopila globiformis, Thiobacillus acidophilus, and Acidiphilium cryptum (level of sequence similarity, 90%). "Erythromicrobium ramosum" and isolate T4T are closely related to Erythrobacter longus and Porphyrobacter neustonensis (level of sequence similarity, 95%). These organisms are members of the alpha-4 subclass of the Proteobacteria. Strain T4T is a motile, red or orange bacterium. The major carotenoids are bacteriorubixanthinal and erythroxanthin sulfate. In vivo measurements revealed bacteriochlorophyll absorption maxima at 377, 590, 800, and 868 nm. Strain T4T grows in the presence of 5 to 96/1000 salinity and uses glucose, fructose, acetate, pyruvate, glutamate, succinate, and lactate as substrates. On the basis of its distinct phylogenetic position and phenotypic characteristics which are different from those of Erythrobacter longus, we propose that strain T4T should be placed in a new species of the genus Erythrobacter, Erythrobacter litoralis. The descriptions of "Roseococcus thiosulfatophilus" and "Erythromicrobium ramosum" are emended.

Phylogenetic Analysis of Bradyrhizobium japonicum and Photosynthetic Stem-Nodulating Bacteria from Aeschynomene Species Grown in Separated Geographical Regions.

Nearly complete and short partial 16S rRNA sequences were derived from PCR-amplified ribosomal DNAs of Bradyrhizobium japonicum USDA 136 and USDA 110 and five strains of bacteriochlorophyll-synthesizing bacteria isolated from stem nodules of Aeschynomene indica and other Aeschynomene species growing in different geographic regions, including India, The Philippines and North America. We confirmed that the five stem-nodulating strains examined synthesize bacteriochlorophyll a, and the absorption spectra of methanol-extracted cells contained a major absorbance peak at 770 nm. Strains isolated on different continents and from different Aeschynomene species were found to be phylogenetically homogeneous and exhibited levels of sequence similarity of more than 99%. The bacteriochlorophyll-synthesizing rhizobia, Bradyrhizobium japonicum, Blastobacter denitrificans, Afipia felis, and Rhodopseudomonas palustris exhibited levels of sequence similarity of 97% or greater and belong to a distinct line of descent within the alpha-2 subdivision of the Proteobacteria. Variable regions between positions 995 and 1045 provide potential target sites for design of a probe that is able to distinguish the photosynthetic rhizobia from closely related taxa.

Porphyrobacter neustonensis gen. nov., sp. nov., an aerobic bacteriochlorophyll-synthesizing budding bacterium from fresh water.

Four strains of orange- or red-pigmented bacteria isolated from freshwater surfaces were shown to synthesize bacteriochlorophyll under aerobic conditions. These strains shared unusual morphological features, such as acellular stalks, crateriformlike structures, and buds, with bacteria in the order Planctomycetales. However, comparisons of 16S rRNA sequences showed them to be members of the alpha-4 subdivision of the class Proteobacteria and most closely related to the marine aerobic bacteriochlorophyll-synthesizing bacterium Erythrobacter longus. They also differ from members of the Planctomycetales phenotypically in their synthesis of bacteriochlorophyll and possession of a peptidoglycan cell wall. They can be distinguished from E. longus on the basis of their 16S rRNA sequence, the G+C content of their DNA, cellular fatty acid composition, and carbon substrate spectrum. A new genus, Porphyrobacter, with a single species, P. neustonensis gen. nov., sp. nov., is proposed for these strains. The type strain is ACM 2844.

Heterotrophic bacteria in an air-handling system.

Heterotrophic bacteria from structural surfaces, drain pan water, and the airstream of a well-maintained air-handling system with no reported building-related illness were enumerated. Visually the system appeared clean, but large populations of bacteria were found on the fin surface of the supply-side cooling coils (10(5) to 10(6) CFU cm-2), in drain pan water (10(5) to 10(7) CFU ml-1), and in the sump water of the evaporative condenser (10(5) CFU ml-1). Representative bacterial colony types recovered from heterotrophic plate count cultures on R2A medium were identified to the genus level. Budding bacteria belonging to the genus Blastobacter dominated the supply surface of the coil fins, the drain pan water, and the postcoil air. These data and independent scanning electron microscopy indicated that a resident population of predominantly Blastobacter bacteria was present as a biofilm on the supply-side cooling coil fins.

Isolation of a bacterium resembling Pirellula species from primary tissue culture of the giant tiger prawn (Penaeus monodon).

During attempts to establish tissue cultures from hepatopancreas, heart, and hemolymph of the giant tiger prawn (Penaeus monodon), using a medium including penicillin, streptomycin, and amphotericin B, bacterial contamination in the form of a sheet of growth attached to the tissue culture vessel was a persistent problem. Contaminant bacteria were teardrop-shaped cells arranged in rosettes, and electron microscopy revealed buds, crateriform structures, and the absence of a peptidoglycan layer in the cell wall, features characteristic of bacteria in the Planctomyces-Pirellula group, a phylogenetically distinct group of eubacteria. Two strains of contaminant bacteria were isolated in pure culture. Both exhibited morphology and antibiotic resistance consistent with their membership in the Planctomyces-Pirellula group (order Planctomycetales) of eubacteria. Tissue culture media for marine invertebrates may select for such bacteria if high concentrations of cell wall synthesis-inhibiting antibiotics are included.

Heterogeneity, persistence, and distribution of Pseudomonas aeruginosa genotypes in cystic fibrosis patients.

A collection of 222 isolates of Pseudomonas aeruginosa was obtained from the respiratory tract of 16 patients with cystic fibrosis over a 4- to 9-month period. Fourteen of these patients were unrelated, while the remaining two were siblings. Isolates were typed by conventional pyocin typing and also by the use of a DNA probe containing 741 bp immediately upstream of the exotoxin A structural gene and the initial 732 bp of the exotoxin A structural gene. By pyocin typing, 69% (11 of 16) of the patients were shown to harbor a single type that persisted in the lung throughout the study. By genotyping (DNA probe typing), all but three patients (13 of 16, 81%) harbored a single persistent genotype in their lungs. Six patients other than the sibling pair (6 of 14, 43%) shared a common genotype in their lungs as judged by DNA probing, and the pyocin type of these isolates was also identical. In four of these six patients, the shared genotype was also the persistent genotype. The sibling pair studied also carried a common genotype in their lungs as indicated by DNA probing, even though the pyocin type of these isolates varied. Results presented suggest that the majority of patients harbor a persistent strain in their lungs and that cross-colonization may occur.

Evaluation of the genus Listonella and reassignment of Listonella damsela (Love et al.) MacDonell and Colwell to the genus Photobacterium as Photobacterium damsela comb. nov. with an emended description.

The genus Listonella, which was recently described on the basis of 5S rRNA sequence data, was found to be of dubious value on the basis of the results of a comparison of a number of taxonomic studies involving members of the Vibrionaceae. The available data suggest that 5S rRNA sequences may be of limited taxonomic use at the intra- and intergeneric levels, at least for apparently recently evolved groups, such as the Vibrionaceae. In this light, we assessed the generic assignment of the species Listonella damsela. Phenotypic characterization of 12 strains of bacteria assigned to L. damsela, including type strain ATCC 33539, revealed a strong resemblance to members of the genus Photobacterium. All of the strains conformed to major characteristics common to all known Photobacterium species. The characteristics of these organisms included the absence of a flagellar sheath and accumulation of poly-beta-hydroxybutyrate during growth on glucose coupled with the inability to utilize DL-beta-hydroxybutyrate as a sole carbon source. On the basis of the phenotypic data, we propose that L. damsela should be reassigned to the genus Photobacterium as Photobacterium damsela comb. nov.

Membrane-bounded nucleoid in the eubacterium Gemmata obscuriglobus.

The freshwater budding eubacterium Gemmata obscuriglobus possesses a DNA-containing nuclear region that is bounded by two nuclear membranes. The membrane-bounded nature of the nucleoid in this bacterium was shown by thin sectioning of chemically fixed cells, thin sectioning of freeze-substituted cells, and freeze-fracture/freeze-etch. The fibrillar nucleoid was surrounded by electron-dense granules that were in turn enveloped by two nuclear membranes separated by an electron-transparent space. Immunogold labeling of thin sections of conventionally fixed cells with anti-double-stranded DNA antibody demonstrated double-stranded DNA associated with fibrillar material within the membrane boundary. The occurrence of a membrane-bounded nucleoid in a eubacterial prokaryote is a significant exception to the evidence supporting the prokaryote/eukaryote dichotomous classification of cell structure.