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S J Giovannoni

Publications and source records attributed to S J Giovannoni.

At least 19 recordsLinked to original sources
PubMed2006

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.

Animals↗
PubMed2004

Prevalence of the Chloroflexi-related SAR202 bacterioplankton cluster throughout the mesopelagic zone and deep ocean.

Since their initial discovery in samples from the north Atlantic Ocean, 16S rRNA genes related to the environmental gene clone cluster known as SAR202 have been recovered from pelagic freshwater, marine sediment, soil, and deep subsurface terrestrial environments. Together, these clones form a major, monophyletic subgroup of the phylum Chloroflexi: While members of this diverse group are consistently identified in the marine environment, there are currently no cultured representatives, and very little is known about their distribution or abundance in the world's oceans. In this study, published and newly identified SAR202-related 16S rRNA gene sequences were used to further resolve the phylogeny of this cluster and to design taxon-specific oligonucleotide probes for fluorescence in situ hybridization. Direct cell counts from the Bermuda Atlantic time series study site in the north Atlantic Ocean, the Hawaii ocean time series site in the central Pacific Ocean, and along the Newport hydroline in eastern Pacific coastal waters showed that SAR202 cluster cells were most abundant below the deep chlorophyll maximum and that they persisted to 3600 m in the Atlantic Ocean and to 4000 m in the Pacific Ocean, the deepest samples used in this study. On average, members of the SAR202 group accounted for 10.2% (+/-5.7%) of all DNA-containing bacterioplankton between 500 and 4000 m.

Atlantic Ocean↗
PubMed2000

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.

Alphaproteobacteria↗
PubMed1999

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%.

Alphaproteobacteria↗
PubMed1999

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.

Anti-Bacterial Agents↗
PubMed1999

Immunochemical detection and isolation of DNA from metabolically active bacteria.

Most techniques used to assay the growth of microbes in natural communities provide no information on the relationship between microbial productivity and community structure. To identify actively growing bacteria, we adapted a technique from immunocytochemistry to detect and selectively isolate DNA from bacteria incorporating bromodeoxyuridine (BrdU), a thymidine analog. In addition, we developed an immunocytochemical protocol to visualize BrdU-labeled microbial cells. Cultured bacteria and natural populations of aquatic bacterioplankton were pulse-labeled with exogenously supplied BrdU. Incorporation of BrdU into microbial DNA was demonstrated in DNA dot blots probed with anti-BrdU monoclonal antibodies and either peroxidase- or Texas red-conjugated secondary antibodies. BrdU-containing DNA was physically separated from unlabeled DNA by using antibody-coated paramagnetic beads, and the identities of bacteria contributing to both purified, BrdU-containing fractions and unfractionated, starting-material DNAs were determined by length heterogeneity PCR (LH-PCR) analysis. BrdU-containing DNA purified from a mixture of DNAs from labeled and unlabeled cultures showed >90-fold enrichment for the labeled bacterial taxon. The LH-PCR profile for BrdU-containing DNA from a labeled, natural microbial community differed from the profile for the community as a whole, demonstrating that BrdU was incorporated by a taxonomic subset of the community. Immunocytochemical detection of cells with BrdU-labeled DNA was accomplished by in situ probing with anti-BrdU monoclonal antibodies and Texas red-labeled secondary antibodies. Using this suite of techniques, microbial cells incorporating BrdU into their newly synthesized DNA can be quantified and the identities of these actively growing cells can be compared to the composition of the microbial community as a whole. Since not all strains tested could incorporate BrdU, these methods may be most useful when used to gain an understanding of the activities of specific species in the context of their microbial community.

Antibodies, Monoclonal↗
PubMed1998

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.

Antarctic Regions↗
PubMed1998

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.

Base Composition↗
PubMed1998

Phylogenetic diversity of ultraplankton plastid small-subunit rRNA genes recovered in environmental nucleic acid samples from the Pacific and Atlantic coasts of the United States.

The scope of marine phytoplankton diversity is uncertain in many respects because, like bacteria, these organisms sometimes lack defining morphological characteristics and can be a challenge to grow in culture. Here, we report the recovery of phylogenetically diverse plastid small-subunit (SSU) rRNA gene (rDNA) clones from natural plankton populations collected in the Pacific Ocean off the mouth of Yaquina Bay, Oreg. (OCS clones), and from the eastern continental shelf of the United States off Cape Hatteras, N.C. (OM clones). SSU rRNA gene clone libraries were prepared by amplifying rDNAs from nucleic acids isolated from plankton samples and cloning them into plasmid vectors. The PCR primers used for amplification reactions were designed to be specific for bacterial SSU rRNA genes; however, plastid genes have a common phylogenetic origin with bacteria and were common in both SSU rRNA gene clone libraries. A combination of restriction fragment length polymorphism analyses, nucleic acid sequencing, and taxon-specific oligonucleotide probe hybridizations revealed that 54 of the 116 OCS gene clones were of plastid origin. Collectively, clones from the OCS and OM libraries formed at least eight unique lineages within the plastid radiation, including gene lineages related to the classes Bacillariophyceae, Cryptophyceae, Prymnesiophyceae, Chrysophyceae, and Prasinophyceae; for a number of unique clones, no close phylogenetic neighbors could be identified with confidence. Only a group of two OCS rRNA gene clones showed close identity to the plastid SSU rRNA gene sequence of a cultured organism [Emiliania huxleyi (Lohmann) Hay and Mohler; 99.8% similar]. The remaining clones could not be identified to the genus or species level. Although cryptic species are not as prevalent among phytoplankton as they are among their bacterial counterparts, this genetic survey nonetheless uncovered significant new information about phytoplankton diversity.

Animals↗
PubMed1998

Screening of a fosmid library of marine environmental genomic DNA fragments reveals four clones related to members of the order Planctomycetales.

A fosmid library with inserts containing approximately 40 kb of marine bacterial DNA (J. L. Stein, T. L. Marsh, K. Y. Wu, H. Shizuya, and E. F. DeLong, J. Bacteriol. 178:591-599, 1996) yielded four clones with 16S rRNA genes from the order Planctomycetales. Three of the clones belong to the Pirellula group and one clone belongs to the Planctomyces group, based on phylogenetic and signature nucleotide analyses of full-length 16S rRNA genes. Sequence analysis of the ends of the genes revealed a consistent mismatch in a widely used bacterium-specific 16S rRNA PCR amplification priming site (27F), which has also been reported in some thermophiles and spirochetes.

Bacteria↗
PubMed1997

Diversity and depth-specific distribution of SAR11 cluster rRNA genes from marine planktonic bacteria.

Small-subunit (SSU) ribosomal DNA (rDNA) gene clusters are phylogenetically related sets of SSU rRNA genes, commonly encountered in genes amplified from natural populations. Genetic variability in gene clusters could result from artifacts (polymerase error or PCR chimera formation), microevolution (variation among rrn copies within strains), or macroevolution (genetic divergence correlated with long-term evolutionary divergence). To better understand gene clusters this study assessed genetic diversity and distribution of a single environmental SSU rDNA gene cluster, the SAR11 cluster. SAR11 cluster genes, from an uncultured group of the alpha subclass of the class Proteobacteria, have been recovered from coastal and midoceanic waters of the North Atlantic and Pacific. We cloned and bidirectionally sequenced 23 new SAR11 cluster 16S rRNA genes, from 80 and 250 m in the Sargasso Sea and from surface coastal waters of the Atlantic and Pacific, and analyzed them with previously published sequences. Two SAR11 genes were obviously PCR chimeras, but the biological (nonchimeric) origins of most subgroups within the cluster were confirmed by independent recovery from separate gene libraries. Using group-specific oligonucleotide probes, we analyzed depth profiles of nucleic acids, targeting both amplified rDNAs and bulk RNAs. Two subgroups within the SAR11 cluster showed different highly depth-specific distributions. We conclude that some of the genetic diversity within the SAR11 gene cluster represents macroevolutionary divergence correlated with niche specialization. Furthermore, we demonstrate the utility for marine microbial ecology of oligonucleotide probes based on gene sequences amplified from natural populations and show that a detailed knowledge of sequence variability may be needed to effectively design these probes.

Animals↗
PubMed1997

The ldh phylogeny for environmental isolates of Lactococcus lactis is consistent with rRNA genotypes but not with phenotypes.

Lactate dehydrogenase (ldh) gene sequences, levels of 16S rRNA group-specific probe binding, and phenotypic characteristics were compared for 45 environmental isolates and four commercial starter strains of Lactococcus lactis to identify evolutionary groups best suited to cheddar cheese manufacture, ldh sequences from the environmental isolates showed high similarity to those from two groups of L. lactis used for industrial fermentations, L. lactis subsp. cremoris and subsp. lactis. Within each phylogenetically defined subspecies, ldh sequence similarities were greater than 99.1%. Strains with phenotypic traits formerly diagnostic for both subspecies were found in each ldh similarity group, but only strains belonging to L. lactis subsp. cremoris by both the newer, genetic and the older, superseded phenotypic criteria were judged potentially suitable for the commercial production of cheddar cheese. Identical evolutionary relationships were inferred from ldh sequences and from binding of subspecies-specific, 16S rRNA-directed oligonucleotide probes. However, groups defined according to these chromosomal traits bore no relationship to patterns of arginine deamination, carbon substrate utilization, or bacteriophage sensitivity, which may be encoded by cryptic genes or sexually transmissible genetic elements. Fourteen new L. lactis subsp. cremoris isolates were identified as suitable candidates for cheddar cheese manufacture, and 10 of these were completely resistant to three different batteries of commercial bacteriophages known to reduce starter activity.

Bacterial Typing Techniques↗
PubMed1997

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.

Bacteria↗
PubMed1997

Bacterial diversity among small-subunit rRNA gene clones and cellular isolates from the same seawater sample.

Numerous investigations applying the cloning and sequencing of rRNA genes (rDNAs) to the study of marine bacterioplankton diversity have shown that the sequences of genes cloned directly from environmental DNA do not correspond to the genes of cultured marine taxa. These results have been interpreted as support for the hypothesis that the most abundant heterotrophic marine bacterioplankton species are not readily culturable by commonly used methods. However, an alternative explanation is that marine bacterioplankton can be easily cultured but are not well represented in sequence databases. To further examine this question, we compared the small-subunit (SSU) rDNAs of 127 cellular clones isolated from a water sample collected off the Oregon coast to 58 bacterial SSU rDNAs cloned from environmental DNAs from the same water sample. The results revealed little overlap between partial SSU rDNA sequences from the cellular clones and the environmental clone library. An exception was the SSU rDNA sequence recovered from a cellular clone belonging to the Pseudomonas subgroup of the gamma subclass of the class Proteobacteria, which was related to a single gene cloned directly from the same water sample (OCS181) (similarity, 94.6%). In addition, partial SSU rDNA sequences from three of the cultured strains matched a novel rDNA clone related to the gamma subclass of the Proteobacteria found previously in an environmental clone library from marine aggregates (AGG53) (similarity, 94.3 to 99.6%). Our results support the hypothesis that many of the most abundant bacterioplankton species are not readily culturable by standard methods but also show that heterotrophic bacterioplankton that are culturable on media with high organic contents include many strains for which SSU rDNA sequences are not available in sequence databases.

Bacteria↗
PubMed1997

A novel delta-subdivision proteobacterial lineage from the lower ocean surface layer.

A small-subunit ribosomal RNA (16S rRNA) gene lineage (SAR324) affiliated with the delta-subdivision of the class Proteobacteria (DP) was discovered in a 16S rRNA gene clone library prepared from a water sample collected from 250 m in the western Sargasso Sea. This clone library of nearly full-length amplicons of bacterial 16S rRNA genes has been the subject of previous studies aimed at identifying bacteria that inhibit the lower ocean surface layer. The novel lineage was identified by randomly sequencing clones that did not hybridize to oligonucleotide probes specific for several abundant bacterioplankton groups identified in previous studies. Phylogenetic analysis indicated that SAR324 was most closely affiliated with the DP, although it showed no specific relationship to any DP 16S rRNA genes in databases. Eight of the clones in the library of 148 clones were identified as members of the SAR324 lineage by hybridization to an oligonucleotide probe specific for SAR324. Subsequent hybridizations showed that the SAR324 group is stratified in the lower surface layer of both the Atlantic and Pacific Oceans, with maxima between 160 and 500 m. The repeated discovery of sequences belonging to different gene clusters with similar distributions in this region of the water column suggests that microbial communities in the lower surface layer may be functionally specialized.

Atlantic Ocean↗
PubMed1996

16S rRNA genes reveal stratified open ocean bacterioplankton populations related to the Green Non-Sulfur bacteria.

Microorganisms play an important role in the biogeochemistry of the ocean surface layer, but spatial and temporal structures in the distributions of specific bacterioplankton species are largely unexplored, with the exceptions of those organisms that can be detected by either autofluorescence or culture methods. The use of rRNA genes as genetic markers provides a tool by which patterns in the growth, distribution, and activity of abundant bacterioplankton species can be studied regardless of the ease with which they can be cultured. Here we report an unusual cluster of related 16S rRNA genes (SAR202, SAR263, SAR279, SAR287, SAR293, SAR307) cloned from seawater collected at 250 m in the Sargasso Sea in August 1991, when the water column was highly stratified and the deep chlorophyll maximum was located at a depth of 120 m. Phylogenetic analysis and an unusual 15-bp deletion confirmed that the genes were related to the Green Non-Sulfur phylum of the domain Bacteria. This is the first evidence that representatives of this phylum occur in the open ocean. Oligonucleotide probes were used to examine the distribution of the SAR202 gene cluster in vertical profiles (0-250 m) from the Atlantic and Pacific Oceans, and in discrete (monthly) time series (O and 200 m) (over 30 consecutive months in the Western Sargasso Sea. The data provide robust statistical support for the conclusion that the SAR202 gene cluster is proportionately most abundant at the lower boundary of the deep chlorophyll maximum (P = 2.33 x 10(-5)). These results suggest that previously unsuspected stratification of microbial populations may be a significant factor in the ecology of the ocean surface layer.

Animals↗
PubMed1996

The marine bacterium Pseudoalteromonas haloplanktis has a complex genome structure composed of two separate genetic units.

The genome size of Pseudoalteromonas haloplanktis, a ubiquitous and easily cultured marine bacterium, was measured as a step toward estimating the genome complexity of marine bacterioplankton. To determine total genome size, we digested P. haloplanktis DNA with the restriction endonucleases Notl and Sfil, separated the fragments using pulsed-field gel electrophoresis (PFGE), and summed the sizes of the fragments. The P. haloplanktis genome was 3512 +/- 112 kb by Notl digestion and 3468 +/- 54.1 kb by Sfil digestion. P. haloplanktis is also shown to have a complex genome structure, composed of two large replicons of approximately 2700 and 800 kb. Three pieces of evidence support this conclusion: (1) Two separate bands are always seen in PFGE of undigested P. haloplanktis DNA; (2) restriction digests of the larger band are missing a band of approximately 650 kb compared with restriction digests of total genomic DNA; and (3) a 16S rDNA probe hybridized to the larger replicon but not to the smaller. To our knowledge, P. haloplanktis is the first marine bacterium shown to have a complex genome structure.

DNA, Bacterial↗