Isolation and growth of a bacterium able to degrade nitrilotriacetic acid under denitrifying conditions

A Gram-negative bacterium was isolated from river sediment which was able to grow with nitrilotriacetic acid as a combined carbon, nitrogen and energy source in the absence of molecular oxygen using nitrate as the terminal electron acceptor. Batch growth parameters and mass balances are reported for growth under both aerobic and denitrifying conditions.The strain was characterized with respect to its substrate spectrum and other physiological properties. This denitrifying isolate is serologically unrelated to the comprehensively described Gram-negative obligately aerobic NTA-degrading bacteria all of which belong to the -subclass of Proteobacteria. Chemotaxonomic characterization, which revealed the presence of spermidine as the main polyamine and ubiquinone Q-8, excludes the new isolate from the phylogenetically redefined genus Pseudomonas and indicates a possible location within the -subclass of Proteobacteria close to, but separate from the genus Xanthomonas.     

Erratum to: Bacterial diversity in toxic<Emphasis Type="Italic"> Alexandrium tamarense</Emphasis> blooms off the Orkney Isles and the Firth of Forth

The genetic diversity of the bacterial community associated with Alexandrium tamarense blooms was studied in blooms of the toxic dinoflagellates in the waters around the Orkney Isles and the Firth of Forth (Scotland). For toxin and molecular analysis of the bacterial communities associated with the toxic bloom, water samples were taken in 1998 and 1999 from A. tamarense blooms. The bacterial community structure, as determined by DGGE (denaturing gradient gel electrophoresis) showed clear differences between all three investigated size fractions (dinoflagellate-associated bacteria, attached bacteria and free-living bacteria), with high diversity within each sample. DNA sequence analysis of the dominant and most frequent DGGE bands revealed the dominance of Proteobacteria, mainly of the Roseobacter clade, with similarities of 91–99%. Moreover, DGGE bands occurring at the same position in the gel throughout in most samples corroborate the presence of several specific Proteobacteria of the Roseobacter clade. Overall, 500 bacteria were isolated from the bloom and partly phylogenetically analysed. They were members of two prokaryotic phyla, the Proteobacteria and the Bacteroidetes, related to Proteobacteria of the and subdivisions (Alteromonas, Pseudoalteromonas and Colwellia). All bacteria were tested for the production of sodium channel blocking (SCB) toxins using mouse neuroblastoma assay. No production of SCB toxins was found and high performance liquid chromatography (HPLC) analysis confirmed these results. The content of total paralytic shellfish poisoning (PSP) toxin in the water samples, as measured within the toxic dinoflagellate blooms using HPLC, ranged from 53 to 2191 ng PSP l^–1 in 1998 and from 0 to 478 ng PSP l^–1 in 1999. Changes in PSP toxin content were not accompanied by changes of DGGE band patterns. We therefore presume that the bacterial groups identified in this study were not exclusively associated with toxic A. tamarense, but were generally associated with the phytoplankton.An erratum to this article can be found at Communicated by H.-D. Franke     

Desulfomonile tiedjei gen. nov. and sp. nov., a novel anaerobic,dehalogenating, sulfate-reducing bacterium

An anaerobic, dehalogenating, sulfate-reducing bacterium, strain DCB-1, is described and nutritionally characterized. The bacterium is a Gram-negative, nonmotile, non-sporeforming large rod with an unusual morphological feature which resembles a collar. The microorganism reductively dehalogenates meta substituted halobenzoates and also reduces sulfate, sulfite and thiosulfate as electron acceptors. The bacterium requires nicotinamide, 1,4-naphthoquinone and thiamine for optimal growth in a defined medium. The microorganism can grow autotrophically on H₂:CO₂ with sulfate or thiosulfate as terminal electron acceptors. It can also grow heterotrophically with pyruvate, several methoxybenzoates, formate plus sulfate or benzoate plus sulfate. It ferments pyruvate to acetate and lactate in the absence of other electron acceptors. The bacterium is inhibited by MoO _inf4 ^sup2- or SeO _inf4 ^sup2- as well as tetracycline, chloramphenicol, kanamycin or streptomycin. Cytochrome c₃ and desulfoviridin have been purified from cells grown in defined medium. 16S rRNA sequence analysis indicates the organism is a new genus of sulfate-reducing bacteria in the delta subdivision of the class Proteobacteria. We propose that the strain be named Desulfomonile tiedjei.Non-standard abbreviations PIPES piperazine-N,N-bis[2-ethanesulfonic acid] - MES 2-[N-morpholino]ethanesulfonic acid - TES N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid - HQNO 2-N-heptyl-4-hydroxy-quinoline-N-oxide - CCCP carbonyl-cyanide-m-chlorophenylhydrazine - CM carboxymethyl     

The eubacterial endosymbionts of whiteflies (homoptera: Aleyrodoidea) constitute a lineage distinct from the endosymbionts of aphids and mealybugs

Whiteflies (superfamily Aleyrodoidea) contain eubacterial endosymbionts localized within host cells known as mycetocytes. Sequence analysis of the genes for the 16S rRNA of the endosymbionts ofBemisia tabaci, Siphoninus phillyreae, andTrialeurodes vaporariorum indicates that these organisms are closely related and constitute a distinct lineage within the -subdivision of theProteobacteria. The endosymbionts of whiteflies are unrelated to the endosymbionts of aphids and mealybugs, which are in two separate lineages.     

Characterization of a DMSP-degrading bacterial isolate from the Sargasso Sea

A bacterium which cleaves dimethylsulfoniopropionate (DMSP) to form dimethylsulfide (DMS) was isolated from surface Sargasso Sea water by a DMSP enrichment technique. The isolate, here designated LFR, is a Gram-negative, obligately aerobic, rod-shaped, carotenoid-containing bacterium with a DNA G+C content of 70%. Sequencing and comparison of its 16S ribosomal ribonucleic acid (rRNA) with that of known eubacteria revealed highest similarity (91% unrestricted sequence similarity) to Roseobacter denitrificans (formerly Erythrobacter species strain OCh114), an aerobic, bacteriochlorophyll-containing marine representative of the -Proteobacteria. However, physiological differences between the two bacteria, and the current lack of other characterized close relatives, preclude assignment of strain LFR to the Roseobacter genus. Screening of fifteen characterized marine bacteria revealed only one, Pseudomonas doudoroffii, capable of degrading DMSP to DMS. Strain LFR is deposited with the American Type Culture Collection (ATCC 51258) and 16S rRNA sequence data are available under GenBank accession number 15345.Contribution no. 8337 of the Woods Hole Oceanographic Institution     

Complete genome sequence of the extremely acidophilic methanotroph isolate V4, Methylacidiphilum infernorum, a representative of the bacterial phylum Verrucomicrobia

Background

The phylum Verrucomicrobia is a widespread but poorly characterized bacterial clade. Although cultivation-independent approaches detect representatives of this phylum in a wide range of environments, including soils, seawater, hot springs and human gastrointestinal tract, only few have been isolated in pure culture. We have recently reported cultivation and initial characterization of an extremely acidophilic methanotrophic member of the Verrucomicrobia, strain V4, isolated from the Hell's Gate geothermal area in New Zealand. Similar organisms were independently isolated from geothermal systems in Italy and Russia.

Results

We report the complete genome sequence of strain V4, the first one from a representative of the Verrucomicrobia. Isolate V4, initially named "Methylokorus infernorum" (and recently renamed Methylacidiphilum infernorum) is an autotrophic bacterium with a streamlined genome of ~2.3 Mbp that encodes simple signal transduction pathways and has a limited potential for regulation of gene expression. Central metabolism of M. infernorum was reconstructed almost completely and revealed highly interconnected pathways of autotrophic central metabolism and modifications of C₁-utilization pathways compared to other known methylotrophs. The M. infernorum genome does not encode tubulin, which was previously discovered in bacteria of the genus Prosthecobacter, or close homologs of any other signature eukaryotic proteins. Phylogenetic analysis of ribosomal proteins and RNA polymerase subunits unequivocally supports grouping Planctomycetes, Verrucomicrobia and Chlamydiae into a single clade, the PVC superphylum, despite dramatically different gene content in members of these three groups. Comparative-genomic analysis suggests that evolution of the M. infernorum lineage involved extensive horizontal gene exchange with a variety of bacteria. The genome of M. infernorum shows apparent adaptations for existence under extremely acidic conditions including a major upward shift in the isoelectric points of proteins.

Conclusion

The results of genome analysis of M. infernorum support the monophyly of the PVC superphylum. M. infernorum possesses a streamlined genome but seems to have acquired numerous genes including those for enzymes of methylotrophic pathways via horizontal gene transfer, in particular, from Proteobacteria.

Reviewers

This article was reviewed by John A. Fuerst, Ludmila Chistoserdova, and Radhey S. Gupta.     

Microbiological assessment of a disease outbreak on corals from Magnetic Island (Great Barrier Reef, Australia)

Unusual disease lesions were observed in Montipora corals on the fringing reef of Magnetic Island (Great Barrier Reef, Australia) following a period of high water temperature in early January 2002. Tissue death in Montipora spp. appeared as a black layer that spread rapidly across the colony surface, though this appeared as the final phase of disease progression (with three previous disease phases now identified, S. Anthony, unpublished). Culture and molecular-based microbial analysis of this layer did not identify a likely microbial pathogen. Despite this, DNA sequencing of microbial 16S rDNA indicated a shift in the bacterial population associated with affected coral tissue. A clone library of the healthy coral sample predominantly contained sequences within the -Proteobacteria. A disease coral sample representing the margin of the black lesion and healthy coral tissue was dominated by sequences, which demonstrated low sequence identity to a range of -Proteobacteria, -Proteobacteria and cyanobacteria. The microbes identified in the diseased Montipora spp. samples are likely to be opportunistic rather than the causative agent of the observed lesion. Studies are in progress to further characterise the ecology of this disease and describe the potential microbial pathogen(s).     

Phylogenetic relationships among the ciliate arthrodontous mosses: Evidence from chloroplast and nuclear DNA sequences

Parsimony and maximum likelihood analyses of combinedtrnL (UAA) 5 exon —trnF (GAA) andrps4 exon cpDNA, and 18S nrDNA sequences of 60 arthrodontous moss taxa indicate strong support for the monophyly of a clade containing theSplachnineae, Orthotrichineae, and diplolepideous alternate sub-orders. A clade including theSplachnineae, Meesiaceae andLeptobryum (Bryaceae) is similarly well supported and forms the sister group to a clade comprising theOrthotrichineae and the other diplolepideous alternate mosses. Within this latter clade a number of well supported lineages are identified, but relationships among these remain poorly resolved. These analyses indicate that the Splachnaceous and Orthotrichaceous peristomes have been independently derived from an ancestral perfect bryoid peristome.     

Molecular Characterization and In Situ Localization of Endosymbiotic 16S Ribosomal RNA and RuBisCO Genes in the Pogonophoran Tissue

Gutless pogonophorans are generally thought to live in symbiosis with methane-oxidizing bacteria (methanotrophs). We identified a 16S ribosomal RNA gene (rDNA) and a ribulose-1,5-bisphosphate carboxlase/oxygenase (RuBisCO, E.C.4.1.1.39) gene that encode the form I large subunit (cbbL) from symbiont-bearing tissue of the pogonophoran Oligobrachia mashikoi. Phylogenetic analysis of the 16S rDNA sequence suggested that the pogonophoran endosymbiont belonged to the -subdivision of Proteobacteria. The endosymbiont was most closely related to an uncultured bacterium from a hydrocarbon seep, forming a unique clade adjacent to the known methanotrophic 16S rDNA cluster. The RuBisCO gene from the pogonophoran tissue was closely related to those of the chemoautotrophic genera Thiobacillus and Hydrogenovibrio. Presence of the RuBisCO gene suggested a methanotrophic symbiosis because some methanotrophic bacteria are known to be capable of autotrophy via the Calvin cycle. In contrast, particulate and soluble methane monooxygenase genes (pmoA and mmoX) and the methanol dehydrogenase gene (mxaF), which are indicators for methanotrophs or methylotrophs, were not detected by repeated trial of polymerase chain reaction. For 16S rRNA and RuBisCO genes, endosymbiotic localizations were confirmed by in situ hybridization. These results support the possibilities that the pogonophoran host has a novel endosymbiont which belongs to the -subdivision of Proteobacteria, and that the endosymbiont has the gene of the autotrophic enzyme RuBisCO.     

Phylogenomic analysis of Odyssella thessalonicensis fortifies the common origin of Rickettsiales, Pelagibacter ubique and Reclimonas americana mitochondrion

Background

The evolution of the Alphaproteobacteria and origin of the mitochondria are topics of considerable debate. Most studies have placed the mitochondria ancestor within the Rickettsiales order. Ten years ago, the bacterium Odyssella thessalonicensis was isolated from Acanthamoeba spp., and the 16S rDNA phylogeny placed it within the Rickettsiales. Recently, the whole genome of O. thessalonicensis has been sequenced, and 16S rDNA phylogeny and more robust and accurate phylogenomic analyses have been performed with 65 highly conserved proteins.

Methodology/Principal Findings

The results suggested that the O. thessalonicensis emerged between the Rickettsiales and other Alphaproteobacteria. The mitochondrial proteins of the Reclinomonas americana have been used to locate the phylogenetic position of the mitochondrion ancestor within the Alphaproteobacteria tree. Using the K tree score method, nine mitochondrion-encoded proteins, whose phylogenies were congruent with the Alphaproteobacteria phylogenomic tree, have been selected and concatenated for Bayesian and Maximum Likelihood phylogenies. The Reclinomonas americana mitochondrion is a sister taxon to the free-living bacteria Candidatus Pelagibacter ubique, and together, they form a clade that is deeply rooted in the Rickettsiales clade.

Conclusions/Significance

The Reclinomonas americana mitochondrion phylogenomic study confirmed that mitochondria emerged deeply in the Rickettsiales clade and that they are closely related to Candidatus Pelagibacter ubique.     

Erratum to Bacterial diversity in toxic<Emphasis Type="Italic"> Alexandrium tamarense</Emphasis> blooms off the Orkney Isles and the Firth of Forth

The genetic diversity of the bacterial community associated with Alexandrium tamarense blooms was studied in blooms of the toxic dinoflagellates in the waters around the Orkney Isles and the Firth of Forth (Scotland). For toxin and molecular analysis of the bacterial communities associated with the toxic bloom, water samples were taken in 1998 and 1999 from A. tamarense blooms. The bacterial community structure, as determined by DGGE (denaturing gradient gel electrophoresis) showed clear differences between all three investigated size fractions (dinoflagellate-associated bacteria, attached bacteria and free-living bacteria), with high diversity within each sample. DNA sequence analysis of the dominant and most frequent DGGE bands revealed the dominance of α Proteobacteria, mainly of the Roseobacter clade, with similarities of 91–99%. Moreover, DGGE bands occurring at the same position in the gel throughout in most samples corroborate the presence of several specific α Proteobacteria of the Roseobacter clade. Overall, 500 bacteria were isolated from the bloom and partly phylogenetically analysed. They were members of two prokaryotic phyla, the Proteobacteria and the Bacteroidetes, related to Proteobacteria of the α and γ subdivisions (Alteromonas, Pseudoalteromonas and Colwellia). All bacteria were tested for the production of sodium channel blocking (SCB) toxins using mouse neuroblastoma assay. No production of SCB toxins was found and high performance liquid chromatography (HPLC) analysis confirmed these results. The content of total paralytic shellfish poisoning (PSP) toxin in the water samples, as measured within the toxic dinoflagellate blooms using HPLC, ranged from 53 to 2191 ng PSP l^?1 in 1998 and from 0 to 478 ng PSP l^?1 in 1999. Changes in PSP toxin content were not accompanied by changes of DGGE band patterns. We therefore presume that the bacterial groups identified in this study were not exclusively associated with toxic A. tamarense, but were generally associated with the phytoplankton.     

Identification of “Candidatus Thioturbo danicus,” a Microaerophilic Bacterium That Builds Conspicuous Veils on Sulfidic Sediments

Molecular analysis of bacteria enriched under in situ-like conditions and mechanically isolated by micromanipulation showed that a hitherto-uncultivated microaerophilic bacterium thriving in oxygen-sulfide counter-gradients (R. Thar and M. Kühl, Appl. Environ. Microbiol. 68:6310-6320, 2000) is affiliated with the -subdivision of the Proteobacteria. The affiliation was confirmed by the use of whole-cell hybridization with newly designed specific oligonucleotide probes. The bacterium belongs to a new genus and received the provisional name “Candidatus Thioturbo danicus.”     

Relationships among the bdellovibrios revealed by partial sequences of 16S ribosomal RNA

Members of the genusBdellovibrio possess the unifying phenotypic trait of attacking and preying upon other Gram-negative bacteria. It has been suggested that this common lifestyle arose by convergent evolution. Physiological and G + C studies have led to the notion that bdellovibrios are a heterogeneous group of loosely related bacteria. We have inferred the phylogenetic relatedness of 12 strains ofBdellovibrio through the analysis of partial 16S ribosomal RNA sequences. Similarity and degree of homology were assessed, and a phylogenetic tree was constructed by the distance matrix method. One branch of the two-branched tree consisted ofB. bacteriovorus and related strains (W, 6-5-S, 109, 109D, 109J, 114, HI Ox9-2, and HI Ox9-3). The other branch was itself branched, withB. starrii, B. stolpii, and marine strain BM4 in separate sub-branches. AllBdellovibrio strains in turn clustered with representatives of the delta division of theProteobacteria. The results indicate that there are at least two subdivisions of the genusBdellovibrio and that present-day bdellovibrios arose from a common ancestor. The placement of the genusBdellovibrio within the delta division of theProteobacteria was confirmed.     

The nucleotide sequence of the puf operon from the purple photosynthetic bacterium, Rhodospirillum molischianum: Comparative analyses of light-harvesting proteins and the cytochrome subunits associated with the reaction centers

The nucleotide sequence of the puf operon of the purple bacterium, Rhodospirillum molischianum, was determined. The operon includes genes coding for the and subunits of the light-harvesting 1 (LH1) complex and the L, M, and cytochrome subunits of the reaction center complex. As in other purple bacteria, the genes are arranged within the operon in this order. As in Rubrivivax gelatinosus, the deduced amino acid sequence of the cytochrome subunit in Rsp. molischianum contains significant deletions at the attachment site to the M subunit compared with that of Rhodopseudomonas viridis. This suggests that the interaction between the cytochrome subunit and the LM core in Rsp. molischianum and Rvi. gelatinosus is different from that in Rps. viridis. Phylogenetic analysis of the light-harvesting proteins indicated that the LH1 and subunits of Rsp. molischianum are included in the lineage of LH1 polypeptides of the purple bacteria, while the LH2 and subunits are positioned apart from LH2 polypeptides of the other purple bacteria together with those of Chromatium vinosum. Based on these phylogenetic analyses, the classification of the light-harvesting proteins in purple bacteria is discussed.     

Correlation ofLegionella pneumophila virulence with the presence of a plasmid

Legionella pneumophila is the causative agent of Legionellosis in man and considered an opportunistic intracellular Gram-negative bacterium that preferentially infects macrophages. The presence of a plasmid in these organisms was determined in cultures of the bacteria grown in vitro. A correlation was observed between the growth of virulent strains of theLegionella in murine macrophages and growth on standard buffered charcoal yeast extract agar supplemented with 0.1% -ketoglutarate (BCYE) agar medium rich in cysteine and widely used for growth of the bacteria in vitro. In contrast, the avirulent isolates of these strains grew well on supplemented Mueller-Hinton (SMH) agar utilized for differentiating virulent from avirulentLegionella. However, one virulent strain ofLegionella (the Iowa strain) was found to grow moderately well on the SMH agar. In addition, test strains ofLegionella that infect in vitro human monocytes were found to grow moderately well on the BCYE- agar, but did not grow on the SMH agar. Examination of these strains for plasmid DNA expression showed that extra chromosomal DNA-containing molecules were present in theL. pneumophila strains characterized as virulent for in vitro growth in macrophages. However, the avirulent strains that replicated in the human monocytes readily but only poorly in the permissive murine macrophages did not show evidence of similar plasmid DNA expression.     

A Bacterium Belonging to the Rickettsiaceae Family Inhabits the Cytoplasm of the Marine Ciliate Diophrys appendiculata (Ciliophora, Hypotrichia)

Bacteria of the family Rickettsiaceae (order Rickettsiales, α-Proteobacteria) are mainly known to be endosymbionts of arthropods with the capability to infect also vertebrate cells. Recently, they have also been found as leech endocytobionts. In the present paper, we report the first finding of a bacterium belonging to the family Rickettsiaceae in a natural population of a marine ciliate protozoan, namely Diophrys appendiculata, collected in the Baltic Sea. Bacteria were unambiguously identified through morphological characterization and the “full-cycle rRNA approach” (i.e., 16S rRNA gene characterization and use of specifically designed oligonucleotide probes for in situ detection). Symbionts are rod-shaped bacteria that grow freely in the cytoplasm of the host cell. They present two different morphotypes, similar in size, but different in cytoplasmic density. These are typical morphological features of members of the family Rickettsiaceae. 16S rRNA gene sequence showed that Diophrys symbionts share a high similarity value (>92%) with bacteria belonging to the genus Rickettsia. Phylogenetic analysis revealed that these new endosymbionts are clearly included in the clade of the family Rickettsiaceae, but they occupy an independent phylogenetic position with respect to members of the genus Rickettsia. This is the first report of a member of this family from a host protozoan and from a marine habitat. This result shows that this bacterial group is more diversified and widespread than supposed so far, and that its ecological relevance could until now have been underestimated. In light of these considerations, the two 16S rRNA oligonucleotide probes here presented, specific for members of the Rickettsiaceae, can represent useful tools for further researches on the presence and the spread of these microorganisms in the natural environment.     

Isolation of the exoelectrogenic denitrifying bacterium Comamonas denitrificans based on dilution to extinction

The anode biofilm in a microbial fuel cell (MFC) is composed of diverse populations of bacteria, many of whose capacities for electricity generation are unknown. To identify functional populations in these exoelectrogenic communities, a culture-dependent approach based on dilution to extinction was combined with culture-independent community analysis. We analyzed the diversity and dynamics of microbial communities in single-chamber air-cathode MFCs with different anode surfaces using denaturing gradient gel electrophoresis based on the 16S rRNA gene. Phylogenetic analyses showed that the bacteria enriched in all reactors belonged primarily to five phylogenetic groups: Firmicutes, Actinobacteria, α-Proteobacteria, β-Proteobacteria, and γ-Proteobacteria. Dilution-to-extinction experiments further demonstrated that Comamonas denitrificans and Clostridium aminobutyricum were dominant members of the community. A pure culture isolated from an anode biofilm after dilution to extinction was identified as C. denitrificans DX-4 based on 16S rRNA sequence and physiological and biochemical characterizations. Strain DX-4 was unable to respire using hydrous Fe(III) oxide but produced 35 mW/m² using acetate as the electron donor in an MFC. Power generation by the facultative C. denitrificans depends on oxygen and MFC configuration, suggesting that a switch of metabolic pathway occurs for extracellular electron transfer by this denitrifying bacterium.     

Prevalence,complete genome,and metabolic potentials of a phylogenetically novel cyanobacterial symbiont in the coral-killing sponge,Terpios hoshinota

Terpios hoshinota is an aggressive, space-competing sponge that kills various stony corals. Outbreaks of this species have led to intense damage to coral reefs in many locations. Here, the first large-scale 16S rRNA gene survey across three oceans revealed that bacteria related to the taxa Prochloron, Endozoicomonas, SAR116, Ruegeria, and unclassified Proteobacteria were prevalent in T. hoshinota. A Prochloron-related bacterium was the most dominant and prevalent cyanobacterium in T. hoshinota. The complete genome of this uncultivated cyanobacterium and pigment analysis demonstrated that it has phycobiliproteins and lacks chlorophyll b, which is inconsistent with the definition of Prochloron. Furthermore, the cyanobacterium was phylogenetically distinct from Prochloron, strongly suggesting that it should be a sister taxon to Prochloron. Therefore, we proposed this symbiotic cyanobacterium as a novel species under the new genus Candidatus Paraprochloron terpiosi. Comparative genomic analyses revealed that ‘Paraprochloron’ and Prochloron exhibit distinct genomic features and DNA replication machinery. We also characterized the metabolic potentials of ‘Paraprochloron terpiosi’ in carbon and nitrogen cycling and propose a model for interactions between it and T. hoshinota. This study builds a foundation for the study of the T. hoshinota microbiome and paves the way for better understanding of ecosystems involving this coral-killing sponge.     

Molecular characterization of meiotic recombination within the major histocompatibility complex of the mouse: Mapping of crossover sites within theI region

The molecular analysis of crossing-over within the mouse major histocompatibility complex provides a useful approach for the study of the structural characteristics of meiotic recombination. In this study five intra-I-region recombinants, each derived fromI ^k/I ^b heterozygotes, were characterized for restriction-fragment length polymorphisms (RFLPs) characteristic of theI region of the two parental strains. Southern blot analysis of intra-I recombinant strains A.TBR2, A.TBR3, A.TBR5, A.TBR13, and A.TBR17 using sixI-region DNA probes revealed that the point of crossing-over in all five recombinants occurred within a 6.2-kbKpnI-EcoRI segment located within theE gene. The segments of DNA containing the crossover point from each of the recombinant chromosomes were cloned by screening partial genomic libraries constructed in gt7 bacteriophage. Construction of partial restriction maps of the cloned segments from the parental and recombinant chromosomes permitted the boundaries of the area containing the crossover site to be narrowed to a 4.0-kb segment located almost entirely within an intron of theE gene. The recognition that the points of crossing-over in all five recombinants studied are clustered in a relatively small area of theI region provides further evidence for a hot spot of recombination associated with theE _ß gene.This work was supported by Grants AI14424 and AI20317 from the National Institutes of Health. J. Kobori was supported by a postdoctoral fellowship from the Arthritis Foundation. E. Zimmerer was supported by a postdoctoral fellowship from the Charles and Johanna Busch Fund of the Bureau of Biological Research. D. Spinella was supported by a predoctoral fellowship from the Charles and Johanna Busch Fund.