The presence of the glycolysis operon in SAR11 genomes is positively correlated with ocean productivity.

Bacteria in the SAR11 clade are highly abundant in marine surface waters, but currently little is known about the carbon compounds that support these large heterotrophic populations. To better understand the carbon requirements of these organisms, we conducted a multiphasic exploration of carbohydrate utilization among SAR11 isolates from the Northeast Pacific Ocean and the Sargasso Sea. A comparison of three SAR11 genomes showed they all lacked a recognizable PTS system, the oxidative portion of the pentose phosphate shunt (zwf-, pgl-), genes for the Embden-Meyerhoff-Parnas (pfk-, pyk-) and Entner-Doudoroff (eda-) pathways of glycolysis. Strain HTCC7211, isolated from an ocean gyre, was missing other glycolysis genes as well. Growth assays, radioisotopes, metagenomics and microarrays were used to test the hypothesis that these isolates might be limited in their abilities to transport and oxidize exogenous carbohydrates. Galactose, fucose, rhamnose, arabinose, ribose and mannose could not serve as carbon sources for the isolates tested. However, differences in glucose utilization were detected between coastal and ocean gyre isolates, with the coastal isolates capable of transporting, incorporating and oxidizing glucose while the open ocean isolate could not. Subsequent microarray analysis of a coastal isolate suggested that an operon encoding a variant of the Entner-Doudoroff pathway is likely responsible for the observed differences in glucose utilization. Metagenomic analysis indicated this operon is more commonly found in coastal environments and is positively correlated with chlorophyll a concentrations. Our results indicated that glycolysis is a variable metabolic property of SAR11 metabolism and suggest that glycolytic SAR11 are more common in productive marine environments.

Molecular phylogeny of the animal kingdom.

A rapid sequencing method for ribosomal RNA was applied to the resolution of evolutionary relationships among Metazoa. Representatives of 22 classes in 10 animal phyla were used to infer phylogenetic relationships, based on evolutionary distances determined from pairwise comparisons of the 18S ribosomal RNA sequences. The classical Eumetazoa are divided into two groups. Cnidarians arose from a protist ancestry different from the second group, the Bilateria. Within the Bilateria, an early split gave rise to Platyhelminthes (flatworms) and the coelomate lineage. Coelomates are thus monophyletic, and they radiated rapidly into four groups: chordates, echinoderms, arthropods, and eucoelomate protostomes.

Perennial Antarctic lake ice: an oasis for life in a polar desert.

The permanent ice covers of Antarctic lakes in the McMurdo Dry Valleys develop liquid water inclusions in response to solar heating of internal aeolian-derived sediments. The ice sediment particles serve as nutrient (inorganic and organic)-enriched microzones for the establishment of a physiologically and ecologically complex microbial consortium capable of contemporaneous photosynthesis, nitrogen fixation, and decomposition. The consortium is capable of physically and chemically establishing and modifying a relatively nutrient- and organic matter-enriched microbial "oasis" embedded in the lake ice cover.

18S rRNA sequences of Leishmania enriettii promastigote and amastigote.

Dideoxy sequencing with reverse transcriptase and universal primers was used to obtain partial sequences of the 18S rRNAs from the promastigote and amastigote life-cycle stages of L. enriettii. Approximately 1400 nucleotides of sequence from the two stages were compared. Unlike Plasmodium berghei, in which 18S rRNAs from the mosquito stage and the mammalian stage of the life cycle are only 96.5% similar, the amastigote and promastigote rRNAs of L. enriettii are identical. In addition, a comparison of 1425 bases of the L. enriettii promastigote sequence with the published sequence of L. donovani revealed only four differences; the two sequences are 99.8% similar. A likely explanation for this high similarity, considering the 97% similarity between L. donovani and the related genus Crithidia fasciculata, is that the two species are closely related and of comparatively recent origin. The low diversity between the 18S rRNA sequences of Leishmania species is similar to that reported for 13 Tetrahymena species, where similarities ranged from 98.1 to 99.9%, but different from the pattern reported in the genus Naegleria, where divergence was greater.

Interspecies recombination between enterococci: genetic and phenotypic diversity of vancomycin-resistant transconjugants.

Handwerger and colleagues demonstrated that a particular clinical isolate of Enterococcus faecium, designated GUC, and here redesignated as GUCR, can conjugatively transfer vancomycin resistance. The vancomycin resistance is encoded by a chromosomally born linked set of genes in the donor, designated the vanA cluster, to the chromosome of an E. faecalis recipient, JH2-2. Here it is reported that an earlier isolate of E. faecium from the same patient who later harbored the vancomycin-resistant E. faecium GUCR lacks the vanA gene cluster but is otherwise similar (by SmaI chromosomal fingerprint and metabolic fingerprinting) to the vancomycin-resistant GUCR. Therefore, "GUCS" is a strong suspect as the base strain for the clinical acquisition of the vanA cluster present in GUCR. Thirteen laboratory-generated vanA transconjugants derived from conjugation between GUCR and JH2-2 were subjected to further analysis, allowing a comparison between transfer in the laboratory and transfer that occurred in the clinical setting. Surprisingly, each JH2-2 transconjugant had a unique constellation of abilities to oxidize various members of a panel of potential carbon sources. This pattern was stable for each transconjugant, and it was not changed by growing the strains with or without vancomycin. Transconjugants had pulsed-field gel electrophoretic (PFGE) patterns largely consistent with that of JH2-2, the recipient in conjugation experiments. However, PFGE analysis showed that a large but variable amount of DNA, between 145 kb and 277 kb, was transferred into different transconjugants. The mechanism appears to be conjugative transposition in which new DNA is added to the pre-existing genome rather than substituting for a segment in the recipient. Mapping and hybridization studies of several transconjugants showed that each received similar, but not exactly the same, DNA fragment of at least 30 kb from the donor. Sequencing of 16S ribosomal genes was used to confirm that the recipient and donor strains used in transconjugation experiments were different species. Sequence analysis was also used to consider the possibility that rRNA operons might be mobilized in conjugation, but no evidence for the transfer of rDNA operons was found. An apparent insertion sequence in E. faecium almost identical to IS 1485 and 57% sequence identity to IS 199 of Streptococcus mutans was found in the region of DNA transferred. The results imply new consequences of conjugative transfer and interspecies recombination.

A PCR fingerprinting technique to distinguish isolates of Lactococcus lactis.

A fingerprinting technique similar to repetitive extragenic palindromic PCR was developed to identify strains of Lactococcus lactis. The method distinguishes closely related strains and discriminates among some with identical ldh sequences. The fingerprinting primer LL-Rep1 complements a moderately repeated sequence found in low G + C Gram-positive bacteria and may therefore prove useful for discriminating among strains of other low G + C Gram-positive species.

A phylogenetic comparison of the 16S rRNA sequence of the fish pathogen, Renibacterium salmoninarum, to gram-positive bacteria.

The 16S rRNA of Renibacterium salmoninarum, the causative agent of bacterial kidney disease in salmonids, was sequenced by reverse transcriptase to produce a nearly complete sequence (97%) of 1475 nucleotides. Phylogenetic comparisons to seventeen genera and signature sequence analysis indicated that R. salmoninarum was a member of the high G + C Gram-positive eubacterial subdivision although the reported G + C value is only 53%. A phylogenetic tree details the relationship of R. salmoninarum to ten actinomycetes from diverse environments.

The Deinococcus-Thermus phylum and the effect of rRNA composition on phylogenetic tree construction.

Through comparative analysis of 16S ribosomal RNA sequences, it can be shown that two seemingly dissimilar types of eubacteria Deinococcus and the ubiquitous hot spring organism Thermus are distantly but specifically related to one another. This confirms an earlier report based upon 16S rRNA oligonucleotide cataloging studies (Hensel et al., 1986). Their two lineages form a distinctive grouping within the eubacteria that deserved the taxonomic status of a phylum. The (partial) sequence of T. aquaticus rRNA appears relatively close to those of other thermophilic eubacteria. e.g. Thermotoga maritima and Thermomicrobium roseum. However, this closeness does not reflect a true evolutionary closeness; rather it is due to a "thermophilic convergence", the result of unusually high G+C composition in the rRNAs of thermophilic bacteria. Unless such compositional biases are taken into account, the branching order and root of phylogenetic trees can be incorrectly inferred.

Phylogenetic analysis of Piscirickettsia salmonis by 16S, internal transcribed spacer (ITS) and 23S ribosomal DNA sequencing.

Piscirickettsia salmonis, the etiologic agent of piscirickettsiosis, is a systemic disease of salmonid fish. Variations in virulence and mortality have been observed during epizootics at different geographical regions and in laboratory experiments with isolates from these different locations. This raises the possibility that biogeographical patterns of genetic variation might be a significant factor with this disease. To assess the genetic variability the 16S ribosomal DNA, the internal transcribed spacer (ITS) and the 23S ribosomal DNA of isolates from 3 different hosts and 3 geographic origins were amplified using the polymerase chain reaction (PCR). Results of this analysis confirm that P. salmonis is a member of the gamma subgroup of the Proteobacteria and show that the isolates form a tight monophyletic cluster with 16S rDNA similarities ranging from 99.7 to 98.5%. The ITS regions were 309 base pairs (bp), did not contain tRNA genes, and varied between isolates (95.2 to 99.7% similarity). Two-thirds of the 23S rRNA gene was sequenced from 5 of the isolates, yielding similarities ranging from 97.9 to 99.8%. Phylogenetic trees were constructed based on the 16S rDNA, ITS and 23S rDNA sequence data and compared. The trees were topologically similar, suggesting that the 3 types of molecules provided similar phylogenetic information. Five of the isolates are closely related (> 99.4% 16S rDNA similarity, 99.1% to 99.7% ITS and 99.3 to 99.8% 23S rDNA similarities). The sequence of one Chilean isolate, EM-90, was unique, with 16S rDNA similarities to the other isolates ranging from 98.5 to 98.9%, the ITS from 95.2 to 96.9% and the 23S rDNA from 97.6 to 98.5%.

Dilution-to-extinction culturing of psychrotolerant planktonic bacteria from permanently ice-covered lakes in the McMurdo Dry Valleys, Antarctica.

Lakes in the McMurdo Dry Valleys of Antarctica are characterized by a permanent ice cover and little or no anthropogenic influence. Although bacterial cultures have been obtained from these habitats, recent culture-independent studies indicate that the most abundant microbes in these systems are not yet cultivated. By using dilution-to-extinction cultivation methods with sterilized and nutrient-amended lake water as media, we isolated 148 chemotrophic psychrotolerant bacterial cultures from fresh surface water of Lake Fryxell and the east lobe of Lake Bonney and the hypersaline, suboxic bottom water from the west lobes of Lake Bonney. Screening of the 16S ribosomal ribonucleic acid (rRNA) genes of the cultures by restriction fragment length polymorphism (RFLP) yielded 57 putatively pure psychrotolerant, slow growing cultures grouped into 18 clusters. The sequencing of 16S rRNA genes of randomly selected representatives of each RFLP cluster revealed that the corresponding isolates belong to the Alphaproteobacteria (six RFLP patterns), Betaproteobacteria (six RFLP patterns), Bacteroidetes (four RFLP patterns), and Actinobacteria (two RFLP patterns). Phylogenetic analysis of the sequences showed that the vast majority of the isolates were not closely related to previously described species. Thirteen of 18 RFLP patterns shared a 16S ribosomal deoxyribonucleic acid sequence similarity of 97% or less with the closest described species, and four isolates had a sequence similarity of 93% or less with the nearest described species. Phylogenetic analysis showed that these sequences were representatives of deeply branching organisms in the respective phylum. A comparison of the isolates with 16S rRNA clone libraries prepared from the same environments showed substantial overlap, indicating that dilution-to-extinction culturing in natural lake water media can help isolate some of the most abundant organisms in these perennially ice-covered lakes.

Pirellula and OM43 are among the dominant lineages identified in an Oregon coast diatom bloom.

Although bacterioplankton and phytoplankton are generally perceived as closely linked in marine systems, specific interactions between discrete bacterioplankton and phytoplankton populations are largely unknown. However, measurements of bacterioplankton distributions during phytoplankton blooms may indicate specific microbial lineages that are responding to phytoplankton populations, and potentially controlling them by producing allelopathic compounds. Here we use a comprehensive molecular approach to identify, characterize and quantify bacterioplankton community responses to an Oregon coast diatom bloom. Total DAPI counts increased by nearly sevenfold in bloom samples, reaching 5.7 x 10(9) cells l(-1), and lineage-specific cell counts using fluorescence in situ hybridization (FISH) indicated that Bacteria accounted for approximately 89% of observed increases. Several dominant members of the bacterial community present outside the bloom (SAR11 and SAR86) did not contribute significantly to observed increases in bloom samples. Clone library and FISH data indicated that uncultured planctomycetes most closely related to Pirellula, and members of the OM43 clade of beta proteobacteria, reached 0.5 x 10(8) and 1.2 x 10(8) cells l(-1), respectively, and were among the dominant lineages in bloom samples.

Transovarial inheritance of endosymbiotic bacteria in clams inhabiting deep-sea hydrothermal vents and cold seeps.

Vesicomyid clams are conspicuous fauna at many deep-sea hydrothermal-vent and cold-seep habitats. All species examined have specialized gill tissue harboring endosymbiotic bacteria, which are thought to provide the hosts' sole nutritional support. In these species mechanisms of symbiont inheritance are likely to be key elements of dispersal strategies. These mechanisms have remained unresolved because the early life stages are not available for developmental studies. A specific 16S rRNA-directed oligodeoxynucleotide probe (CG1255R) for the vesocomyid endosymbionts was used in a combination of sensitive hybridization techniques to detect and localize the endosymbionts in host germ tissues. Symbiont-specific polymerase chain reaction amplifications, comparative gene sequencing, and restriction fragment length polymorphisms were used to detect and confirm the presence of symbiont target in tissue nucleic acid extracts. Nonradioactive in situ hybridizations were used to resolve the position of the bacterial endosymbionts in host cells. Symbiont 16S rRNA genes were consistently amplified from the ovarial tissue of three species of vesicomyid clams: Calyptogena magnifica, C. phaseoliformis, and C. pacifica. The nucleotide sequences of the genes amplified from ovaries were identical to those from the respective host symbionts. In situ hybridizations to CG1255R labeled with digoxigenin-11-dUTP were performed on ovarial tissue from each of the vesicomyid clams. Detection of hybrids localized the symbionts to follicle cells surrounding the primary oocytes. These results suggest that vesicomyid clams assure successful, host-specific inoculation of all progeny by using a transovarial mechanism of symbiont transmission.

Detection of stratified microbial populations related to Chlorobium and Fibrobacter species in the Atlantic and Pacific oceans.

A gene lineage (SAR406) related to Chlorobium and Fibrobacter species was found in 16S rRNA gene clone libraries prepared from samples from two oceans. The clone libraries were constructed from total picoplankton genomic DNA to assess bacterial diversity in the lower surface layer. The samples were collected by filtration from a depth of 80 m at a site in the western Sargasso Sea and from a depth of 120 m at a site in the Pacific Ocean, approximately 70 km from the Oregon coast. The PCR and primers which amplified nearly full-length 16S rRNA genes were used to prepare the clone libraries. Among the diverse gene clones in these libraries were two related clones (SAR406 and OCS307) which could not be assigned to any of the major bacterial phyla. Phylogenetic analyses demonstrated that these genes were distant relatives of the genus Fibrobacter and the green sulfur bacterial phylum, which includes the genus Chlorobium. The inclusion of SAR406 in phylogenetic trees inferred by several methods resulted in support from bootstrap replicates for the conclusion that Fibrobacter and Chlorobium species and SAR406 are a monophyletic group. An oligonucleotide probe that selectively hybridized to clone SAR406 was used to examine the distribution of this gene lineage in vertical profiles from the Atlantic and Pacific Oceans and in monthly time series at 0 and 200 m in the Atlantic Ocean. During stratified periods, the genes were most abundant slightly below the deep chlorophyll layer. Seasonal changes in the surface abundance of SAR406 rDNA were highly correlated with chlorophyll a levels (r = 0.75).

A red Beneckea from Laguna Figueroa, Baja California.

A new bacterium (nitrate-respiring, prodigiosin-producing, marine curved rod with a sheathed flagellum) has been isolated from anaerobic mud underlying a microbial meat. This brightly pigmented red bacterium, referred to as strain BV1 (Baja California vibrio, isolate 1) was taken from a closed, hypersaline basin at Laguna Figueroa (or Laguna Mormona), Baja California de Norte, Mexico. It is closely related to the recently described Beneckea gazogenes (Harwood, 1978), which was isolated from an estuarine habitat, the Sippewissett salt marsh at Woods Hole, Massachusetts, U.S.A. Strain BV1 and B-gazogenes are both oxidase positive facultative anaerobic curved rods which bear a single polar flagellum, and synthesize the red-orange tri-pyrrole pigment prodigiosin. The bacterium, which fluoresces green when excited with UV light (lambda = 455 nm), deposits pigment extracellularly in copious quantities. The extracellular pigment deposits fluoresce red-yellow. Both BV1 and B. gazogenes are able to grow utilizing xylose, cellobiose or arabinose, products of plant biosynthesis, as sole carbon sources. BV1 differs from B. gazogenes in cell size, pattern of pigment production, nutritional characteristics, the ability to perform anaerobic respiration using nitrate as a terminal electron acceptor, sensitivity to a newly discovered lytic phage and to the antibiotic vibriostat O/129.

Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR.

The PCR is used widely for the study of rRNA genes amplified from mixed microbial populations. These studies resemble quantitative applications of PCR in that the templates are mixtures of homologs and the relative abundance of amplicons is thought to provide some measure of the gene ratios in the starting mixture. Although such studies have established the presence of novel rRNA genes in many natural ecosystems, inferences about gene abundance have been limited by uncertainties about the relative efficiency of gene amplification in the PCR. To address this question, three rRNA gene standards were prepared by PCR, mixed in known proportions, and amplified a second time by using primer pairs in which one primer was labeled with a fluorescent nucleotide derivative. The PCR products were digested with restriction endonucleases, and the frequencies of genes in the products were determined by electrophoresis on an Applied Biosystems 373A automated DNA sequencer in Genescan mode. Mixtures of two templates amplified with the 519F-1406R primer pair yielded products in the predicted proportions. A second primer pair (27F-338R) resulted in strong bias towards 1:1 mixtures of genes in final products, regardless of the initial proportions of the templates. This bias was strongly dependent on the number of cycles of replication. The results fit a kinetic model in which the reannealing of genes progressively inhibits the formation of template-primer hybrids.

Phylogenetic analysis of a natural marine bacterioplankton population by rRNA gene cloning and sequencing.

The identification of the prokaryotic species which constitute marine bacterioplankton communities has been a long-standing problem in marine microbiology. To address this question, we used the polymerase chain reaction to construct and analyze a library of 51 small-subunit (16S) rRNA genes cloned from Sargasso Sea bacterioplankton genomic DNA. Oligonucleotides complementary to conserved regions in the 16S rDNAs of eubacteria were used to direct the synthesis of polymerase chain reaction products, which were then cloned by blunt-end ligation into the phagemid vector pBluescript. Restriction fragment length polymorphisms and hybridizations to oligonucleotide probes for the SAR11 and marine Synechococcus phylogenetic groups indicated the presence of at least seven classes of genes. The sequences of five unique rDNAs were determined completely. In addition to 16S rRNA genes from the marine Synechococcus cluster and the previously identified but uncultivated microbial group, the SAR11 cluster [S. J. Giovannoni, T. B. Britschgi, C. L. Moyer, and K. G. Field. Nature (London) 345:60-63], two new gene classes were observed. Phylogenetic comparisons indicated that these belonged to unknown species of alpha- and gamma-proteobacteria. The data confirm the earlier conclusion that a majority of planktonic bacteria are new species previously unrecognized by bacteriologists.

Identification of bacterial cells by chromosomal painting.

Chromosomal painting is a technique for the microscopic localization of genetic material. It has been applied at the subcellular level to identify regions of eukaryotic chromosomes. Here we describe the development of bacterial chromosomal painting (BCP), a related technology for the identification of bacterial cells. Purified genomic DNAs from six bacterial strains were labeled by nick translation with the fluorochrome Fluor-X, Cy3, or Cy5. The average size of the labeled fragments was ca. 50 to 200 bp. The probes were hybridized to formaldehyde-fixed microbial cells attached to slides and visualized by fluorescence microscopy. In reciprocal comparisons, distantly related members of the class Proteobacteria (Escherichia coli and Oceanospirillum linum), different species of the genus Bacillus (B. subtilis and B. megaterium), and different serotypes of the subspecies Salmonella choleraesuis subsp. choleraesuis (serotype typhimurium LT2 and serotype typhi Ty2) could easily be distinguished. A combination of two probes, each labeled with a different fluorochrome, was used successfully to simultaneously identify two cell types in a mixture. Lysozyme treatment was required for the identification of Bacillus spp., and RNase digestion and pepsin digestion were found to enhance signal strength and specificity for all cell types tested. Chromosome in situ suppression, a technique that removes cross-hybridizing fragments from the probe, was necessary for the differentiation of the Salmonella serotypes but was not required to distinguish the more distantly related taxa. BCP may have applications in diverse branches of microbiology where the objective is the identification of bacterial cells.

Bacterial chromosomal painting for in situ monitoring of cultured marine bacteria.

We previously described a new method, bacterial chromosomal painting (BCP), for the in situ identification of bacterial cells. Here, we describe the application of this technique to study the ecology and physiology of cultured marine pelagic bacteria from the western Sargasso Sea (WSS). A total of 86 bacteria were isolated from seawater collected from near the surface, at a depth of 250 m and from nutrient-amended seawater incubations. The 10 bacterial isolates that were best represented in environmental genomic DNA from the WSS were selected using reverse genome probing. BCP hybridization cell counts were used to determine the depth-specific distribution of one of the alpha proteobacterial isolates, B5-6, in the WSS during two thermal stratification regimes: stratified and partially mixed. The maximum cell count measured for B5-6 at the summer deep chlorophyll maximum was approximately 4% of the total cell count. This study is the first application of BCP to natural environments.