Seasonal and spatial variability in Lake Michigan sediment small-subunit rRNA concentrations.

We have used molecular biological methods to study the distribution of microbial small-subunit rRNAs (SSU rRNAs), in relation to chemical profiles, in offshore Lake Michigan sediments. The sampling site is at a depth of 100 m, with temperatures of 2 to 4 degrees C year-round. RNA extracted from sediment was probed with radiolabeled oligonucleotides targeting bacterial, archaeal, and eukaryotic SSU rRNAs, as well as with a universal probe. The coverage of these probes in relation to the present sequence database is discussed. Because ribosome production is growth rate regulated, rRNA concentrations are an indicator of the microbial populations active in situ. Over a 1-year period, changes in sedimentary SSU rRNA concentrations followed seasonal changes in surface water temperature and SSU rRNA concentration. Sedimentary depth profiles of oxygen, reduced manganese and iron, and sulfate changed relatively little from season to season, but the nitrate concentration was approximately fivefold higher in April and June 1997 than at the other times sampling was done. We propose that sediment microbial SSU rRNA concentrations at our sampling site are influenced by seasonal inputs from the water column, particularly the settling of the spring diatom bloom, and that the timing of this input may be modulated by grazers, such that ammonia becomes available to sediment microbes sooner than fresh organic carbon. Nitrate production from ammonia by autotrophic nitrifying bacteria, combined with low activity of heterotrophic denitrifying bacteria in the absence of readily degradable organic carbon, could account for the cooccurrence of high nitrate and low SSU rRNA concentrations.

Microbiological, molecular biological and stable isotopic evidence for nitrogen fixation in the open waters of Lake Michigan.

We have used a combination of microbiological, molecular biological and stable isotope methods to relate specific microbial populations to elemental cycling at an offshore site in Lake Michigan. Several lines of evidence suggest that atmospheric N2 may be a significant source of nitrogen to the lake. Particulate organic nitrogen (PON) at approximately equals 10-15m depth in July and October had a delta15N of 0.5-1.5%o. These values closely reflect the 15N composition of atmospheric N2, suggesting biological nitrogen fixation. Historical data show a developing late-summer N:P minimum at approximately equals 15 m; low abundance of inorganic nitrogen relative to phosphorus favours species able to acquire atmospheric nitrogen. Microscopic examination of October water samples revealed abundant heterocystous cyanobacteria, including Nodularia sp. Potentially nitrogen-fixing Anabaena spp. have been found in Lake Michigan before but, to our knowledge, this is the first report of Nodularia. Finally, we have amplified both cyanobacterial and non-cyanobacterial nifH sequences (encoding the nitrogenase iron protein) from lakewater samples, evidence for the presence of bacteria capable of nitrogen fixation. The surface waters of Lake Michigan are considered to be phosphate limited in the stratified season and, under these conditions, energetically expensive nitrogen fixation is expected to be uncompetitive with assimilation of combined nitrogen. Our results suggest that, from both microbiological and biogeochemical perspectives, this may be an oversimplification.

Distribution and abundance of Gram-positive bacteria in the environment: development of a group-specific probe.

We developed a 16S rRNA-targeted oligonucleotide probe (S-P-GPos-1200-a-A-13) for the Gram-positive bacteria, confirmed its specificity by database searches and hybridization studies, and investigated the effects of humic acids on membrane hybridizations with this probe. S-P-GPos-1200-a-A-13 was used to estimate the abundance of Gram-positive populations in the bovine rumen and Lake Michigan sediments. This probe should be useful for studies of the environmental distribution of Gram-positive bacteria and the detection of uncultured, phylogenetically Gram-positive bacteria with variable or negative Gram staining reactions, and could serve for Gram staining in some diagnostic settings.

Shewanella pealeana sp. nov., a member of the microbial community associated with the accessory nidamental gland of the squid Loligo pealei.

A new, mesophillic, facultatively anaerobic, psychrotolerant bacterium, strain ANG-SQ1T (T = type strain), was isolated from a microbial community colonizing the accessory nidamental gland of the squid Loligo pealei. It was selected from the community on the basis of its ability to reduce elemental sulfur. The cells are motile, Gram-negative rods (2.0-3.0 microns long, 0.4-0.6 micron wide). ANG-SQ1T grows optimally over the temperature range of 25-30 degrees C and a pH range of 6.5-7.5 degrees C in media containing 0.5 M NaCl. 16S rRNA sequence analysis revealed that this organism belongs to the gamma-3 subclass of the Proteobacteria. The closest relative of ANG-SQ1T is Shewanella gelidimarina, with a 16S rRNA sequence similarity of 97.0%. Growth occurs with glucose, lactate, acetate, pyruvate, glutamate, citrate, succinate, Casamino acids, yeast extract or peptone as sole energy source under aerobic conditions. The isolate grows anaerobically by the reduction of iron, manganese, nitrate, fumarate, trimethylamine-N-oxide, thiosulfate or elemental sulfur as terminal electron acceptor with lactate. Growth of ANG-SQ1T was enhanced by the addition of choline chloride to growth media lacking Casamino acids. The addition of leucine or valine also enhanced growth in minimal growth media supplemented with choline. The results of both phenotypic and genetic characterization indicate that ANG-SQ1T is a Shewanella species. Thus it is proposed that this new isolate be assigned to the genus Shewanella and that it should be named Shewanella pealeana sp. nov., in recognition of its association with L. pealei.

Population structure and phylogenetic characterization of marine benthic Archaea in deep-sea sediments.

During the past few years Archaea have been recognized as a widespread and significant component of marine picoplankton assemblages and, more recently, the presence of novel archaeal phylogenetic lineages has been reported in coastal marine benthic environments. We investigated the relative abundance, vertical distribution, phylogenetic composition, and spatial variability of Archaea in deep-sea sediments collected from several stations in the Atlantic Ocean. Quantitative oligonucleotide hybridization experiments indicated that the relative abundance of archaeal 16S rRNA in deep-sea sediments (1500 m deep) ranged from about 2.5 to 8% of the total prokaryotic rRNA. Clone libraries of PCR-amplified archaeal rRNA genes (rDNA) were constructed from 10 depth intervals obtained from sediment cores collected at depths of 1,500, 2,600, and 4,500 m. Phylogenetic analysis of rDNA sequences revealed the presence of a complex archaeal population structure, whose members could be grouped into discrete phylogenetic lineages within the two kingdoms, Crenarchaeota and Euryarchaeota. Comparative denaturing gradient gel electrophoresis profile analysis of archaeal 16S rDNA V3 fragments revealed a significant depth-related variability in the composition of the archaeal population.

Polyphasic taxonomy of the genus Shewanella and description of Shewanella oneidensis sp. nov.

The genus Shewanella has been studied since 1931 with regard to a variety of topics of relevance to both applied and environmental microbiology. Recent years have seen the introduction of a large number of new Shewanella-like isolates, necessitating a coordinated review of the genus. In this work, the phylogenetic relationships among known shewanellae were examined using a battery of morphological, physiological, molecular and chemotaxonomic characterizations. This polyphasic taxonomy takes into account all available phenotypic and genotypic data and integrates them into a consensus classification. Based on information generated from this study and obtained from the literature, a scheme for the identification of Shewanella species has been compiled. Key phenotypic characteristics were sulfur reduction and halophilicity. Fatty acid and quinone profiling were used to impart an additional layer of information. Molecular characterizations employing small-subunit 16S rDNA sequences were at the limits of resolution for the differentiation of species in some cases. As a result, DNA-DNA hybridization and sequence analyses of a more rapidly evolving molecule (gyrB gene) were performed. Species-specific PCR probes were designed for the gyrB gene and used for the rapid screening of closely related strains. With this polyphasic approach, in addition to the ten described Shewanella species, two new species, Shewanella oneidensis and 'Shewanella pealeana', were recognized; Shewanella oneidensis sp. nov. is described here for the first time.

Sulphate reduction and vertical distribution of sulphate-reducing bacteria quantified by rRNA slot-blot hybridization in a coastal marine sediment.

In the past, enumeration of sulphate-reducing bacteria (SRB) by cultivation-based methods generally contradicted measurements of sulphate reduction, suggesting unrealistically high respiration rates per cell. Here, we report evidence that quantification of SRB rRNA by slot-blot hybridization is a valuable tool for a more realistic assessment of SRB abundance in the natural environment. The distribution of SRB was investigated in a coastal marine sediment by hybridization of membrane-immobilized rRNA with oligonucleotide probes. As represented by general probe-target groups, SRB rRNA contributed between 18% and 25% to the prokaryotic rRNA pool. The dominant SRB were related to complete oxidizing genera (Desulphococcus, Desulphosarcina and Desulphobacterium), while Desulphobacter could not be detected. The vertical profile and quantity of rRNA from SRB was compared with sulphate reduction rates (SRR) measured with 35SO4(2-) tracer in whole-core incubations. While SRB abundance was highest near the surface, peaking at around 1.5 cm, measured sulphate reduction rates were lowest in this region. A second peak of SRB rRNA was observed at the transition zone from oxidized to reduced sediment, directly above the sulphate reduction maximum. Cell numbers calculated by converting the relative contribution of SRB rRNA to the percentage of DAPI-stained cells indicated a population size for SRB of 2.4-6.1 x 10(8) cells cm(-3) wet sediment. Cellular sulphate reduction rates calculated on the basis of these estimated cell numbers were between 0.01 and 0.09 fmol SO4(2-) cell(-1) day(-1), which is below the rates that have been determined for pure cultures (0.2-50 fmol SO4(2-) cell(-1) day(-1)) growing exponentially at nearoptimal temperature with a surplus of substrates.

Transcription of the Rhodobacter sphaeroides cycA P1 promoter by alternate RNA polymerase holoenzymes.

These experiments sought to identify what form of RNA polymerase transcribes the P1 promoter for the Rhodobacter sphaeroides cytochrome c2 gene (cycA). In vitro, cycA P1 was recognized by an RNA polymerase holoenzyme fraction that transcribes several well-characterized Escherichia coli heat shock (sigma32) promoters. The in vivo effects of mutations flanking the transcription initiation site (+1) also suggested that cycA P1 was recognized by an RNA polymerase similar to E. coli Esigma32. Function of cycA P1 was not altered by mutations more than 35 bp upstream of position +1 or by alterations downstream of -7. A point mutation at position -34 that is towards the E. coli Esigma32 -35 consensus sequence (G34T) increased cycA P1 activity approximately 20-fold, while several mutations that reduced or abolished promoter function changed highly conserved bases in presumed -10 or -35 elements. In addition, cycA P1 function was retained in mutant promoters with a spacer region as short as 14 nucleotides. When either wild-type or G34T promoters were incubated with reconstituted RNA polymerase holoenzymes, cycA P1 transcription was observed only with samples containing either a 37-kDa subunit that is a member of the heat shock sigma factor family (Esigma37) or a 38-kDa subunit that also allows core RNA polymerase to recognize E. coli heat shock promoters (Esigma38). (R. K. Karls, J. Brooks, P. Rossmeissl, J. Luedke, and T. J. Donohue, J. Bacteriol. 180:10-19, 1998).

Crenarchaeota in Lake Michigan sediment.

RNA from Lake Michigan sediment was hybridized with a DNA probe for archaeal 16S rRNA. There was a peak of archaeal rRNA abundance in the oxic zone and another immediately below it. Six contributing species were identified by PCR amplification of extracted DNA with primers specific for archaeal rDNA: two related to Methanosarcina acetivorans and four related to marine crenarchaeotal sequences. rRNA quantification using a DNA probe specific for this crenarchaeotal assemblage showed it is most abundant in the oxic zone, where it accounts for about 10% of total archaeal rRNA.

Evidence for two promoters for the cytochrome c2 gene (cycA) of Rhodobacter sphaeroides.

Rhodobacter sphaeroides cytochrome c2 (cyt c2) is a periplasmic heme protein, encoded by cycA, that is required for photosynthetic growth and for one branch of the aerobic electron transport chain. cycA mRNA and cyt c2 are more abundant photosynthetically than aerobically. We report here that there are four cycA transcripts by high-resolution Northern (RNA) blot analysis, and we have mapped 10 5' ends by primer extension. Complementation of a cycA null mutant shows that there are at least two cycA promoters: one within 89 bp upstream of the translation initiation codon for a transcript beginning at -28, and at least one within 484 bp upstream for the remaining nine 5' ends. The 5' ends at -28 and -137 are more abundant in aerobically grown cells, while those at -38, -155, -250, and -300 are more abundant photosynthetically. DNA sequences with homology to the Escherichia coli sigma 70 consensus promoter sequence precede the 5' ends at -28 and -274, and there is weak homology upstream of the -82 and -250 ends.

Genetic and physical mapping of the Rhodobacter sphaeroides photosynthetic gene cluster from R-prime pWS2.

Plasmid pWS2 is an R68.45 chimera originally isolated as an R-prime which complemented the Rhodobacter sphaeroides bch-420 allele. Our experiments have shown that pWS2 is also able to complement a wide range of R. sphaeroides pigment and photosynthetic mutants employing nitrosoquanidine, transposon or insertion-generated mutations effecting puhA, puc, puf, cycA, bch, and crt genes. A combination of orthogonal-field-alternation gel electrophoresis, transverse alternating field gel electrophoresis, and conventional electrophoresis have been used to estimate the size of pWS2 at congruent to 168.3 +/- 3.5 kb. A restriction map of the congruent to 109 kb of R. sphaeroides insert DNA was generated by partial and complete restriction endonuclease digestion coupled with Southern hybridization analysis using either gene-specific or junction fragment probes. Genes encoding bacteriochlorophyll (Bchl)-binding proteins (pufBALMX, pucBA, and puhA), cytochrome c2 (cycA), and enzymes involved in Bchl (bch) and carotenoid (crt) biosynthesis have been shown to reside within a contiguous 53-kb region of the R. sphaeroides DNA present on pWS2. The puf operon lies at one end of the 53-kb segment, while the genes puhA, cycA, and pucBA, the latter two of which are located within congruent to 12.0 kb of each other, define the other end of this 53-kb region. The genetic and physical mapping data provided in this paper are discussed in terms of the similarities and differences in the organization of the photosynthetic gene cluster between R. sphaeroides and other photosynthetic bacteria as well as highlighting the use of pWS2 in studies of photosynthetic gene structure and function.

Sarcoid-like disorder of the intracranial optic nerve.

Two cases with a sarcoid-like disorder apparently restricted to the intracranial optic nerve and the adjacent dura mater are described, doublilng the number of reported cases. In both instances there were unilateral visual loss and unusual radiographic changes in the optic canal area. Biopsy samples showed localised infiltration of tissues with non-caseating tubercles containing epithelioid and multinucleated giant cells.

Myelin composition in acute and chronic multiple sclerosis in relation to cerebral lysosomal activity.

The neuropathology of 3 cases of acute multiple sclerosis was correlated with biochemical analyses. Astrocytosis was a characteristic feature of the diffuse demyelinating lesions in one case and lymphocytic cuffing characterized the well-defined plaques present in the white matter of the other two cases. No abnormalities were found in the protein or lipid composition of isolated myelin, despite a wide range of recovery. Nevertheless, the gel electrophoretic protein pattern of white matter adjacent to plaque areas showed selective loss of myelin basic protein. Lysosomal acid proteinase and beta-glucuronidase levels were very significantly increased in all white matter samples in which astrocytosis was a major neuropathological feature. Levels were less markedly raised in samples containing discrete active plaques. Enzyme changes were also found in the apparently normal white matter of 2 of the cases. Acid proteinase activity was in the normal range but the activities of beta-glucuronidase and acetylcholine esterase were elevated. The significance of these results in relation to glial cell activity in the early stages of demyelination is discussed.

Intracranial epithelial cysts. Report of two cases.

The authors report two cases with large unilocular intracerebral epithelial cysts. Diagnosis was facilitated in both patients by computerized tomography (EMI scanner). The clinical and diagnostic aspects of previously reported cases are reviewed, and the etiology and pathogenesis of these cysts discussed.

Benign sinus histiocytosis with massive lymphadenopathy: transient immunological defects in a child with mediastinal involvement.

A 16-month-old boy presented with cervical lymphadenopathy and a mediastinal mass causing tracheal displacement. Treatment of what at first was suspected to be a malignant neoplasm was limited to low-dose irradiation of the mediastinum and biopsy excision of the cervical lymph nodes. There has been no recurrence of disease in the subsequent four years. The pathological features and clinical course correspond to a benign disease first described in 1969 in which there is massive proliferation of histiocytes in lymph node sinuses. No infectious cause was identified. Consistent but eventually reversible defects in lymphocyte response to phytohaemagglutinin and in augmentation of nitro-blue tetrazolium reduction by neutrophils during phagocytosis were demonstrated in the patient and in his monozygous twin. These defects in cellular immune function are believed to be important in the pathogenesis of the histiocytosis.