Formyltetrahydrofolate Synthetase Sequences from Salt Marsh Plant Roots Reveal a Diversity of Acetogenic Bacteria and Other Bacterial Functional Groups

Sixty-two partial formyltetrahydrofolate synthetase (FTHFS) structural gene sequences were recovered from roots of salt marsh plants, including Spartina alterniflora, Salicornia virginica, and Juncus roemerianus. Only S. alterniflora roots yielded sequences grouping with FTHFS sequences from known acetogens. Most other FTHFS or FTHFS-like sequences grouped with those from sulfate-reducing bacteria. Several sequences that grouped with Sphingomonas paucimobilis ligH were also recovered.     

Development of Genomic Tools for the Identification of Certain Pseudomonas up to Species Level

Pseudomonas is a highly versatile bacterium at the species level with great ecological significance. These genetically and metabolically diverse species have undergone repeated taxonomic revisions. We propose a strategy to identify Pseudomonas up to species level, based on the unique features of their 16S rDNA (rrs) gene sequence, such as the frame work of sequences, sequence motifs and restriction endonuclease (RE) digestion patterns. A species specific phylogenetic framework composed of 31 different rrs sequences, allowed us to segregate 1,367 out of 2,985 rrs sequences of this genus, which have been classified at present only up to genus (Pseudomonas) level, as follows: P. aeruginosa (219 sequences), P. fluorescens (463 sequences), P. putida (347 sequences), P. stutzeri (197 sequences), and P. syringae (141 sequences). These segregations were validated by unique 30–50 nucleotide long motifs and RE digestion patterns in their rrs. A single gene thus provides multiple makers for identification and surveillance of Pseudomonas.     

A general method for fast multiple sequence alignment

We have developed a fast heuristic algorithm for multiple sequence alignment which provides near-to-optimal results for sufficiently homologous sequences. The algorithm makes use of the standard dynamic programming procedure by applying it to all pairs of sequences. The resulting score matrices for pair-wise alignment give rise to secondary matrices containing the additional charges imposed by forcing the alignment path to run through a particular vertex. Such a constraint corresponds to slicing the sequences at the positions defining that vertex, and aligning the remaining pairs of prefix and suffix sequences separately. From these secondary matrices, one can compute - for any given family of sequences - suitable positions for cutting all of these sequences simultaneously, thus reducing the problem of aligning a family of n sequences of average length l in a Divide and Conquer fashion to aligning two families of n sequences of approximately half that length.In this paper, we explain the method for the case of 3 sequences in detail, and we demonstrate its potential and its limits by discussing its behaviour for several test families. A generalization for aligning more than 3 sequences is lined out, and some actual alignments constructed by our algorithm for various user-defined parameters are presented.     

Host Distributions of Uncultivated Fecal Bacteroidales Bacteria Reveal Genetic Markers for Fecal Source Identification

The purpose of this study was to examine host distribution patterns among fecal bacteria in the order Bacteroidales, with the goal of using endemic sequences as markers for fecal source identification in aquatic environments. We analyzed Bacteroidales 16S rRNA gene sequences from the feces of eight hosts: human, bovine, pig, horse, dog, cat, gull, and elk. Recovered sequences did not match database sequences, indicating high levels of uncultivated diversity. The analysis revealed both endemic and cosmopolitan distributions among the eight hosts. Ruminant, pig, and horse sequences tended to form host- or host group-specific clusters in a phylogenetic tree, while human, dog, cat, and gull sequences clustered together almost exclusively. Many of the human, dog, cat, and gull sequences fell within a large branch containing cultivated species from the genus Bacteroides. Most of the cultivated Bacteroides species had very close matches with multiple hosts and thus may not be useful targets for fecal source identification. A large branch containing cultivated members of the genus Prevotella included cloned sequences that were not closely related to cultivated Prevotella species. Most ruminant sequences formed clusters separate from the branches containing Bacteroides and Prevotella species. Host-specific sequences were identified for pigs and horses and were used to design PCR primers to identify pig and horse sources of fecal pollution in water. The primers successfully amplified fecal DNAs from their target hosts and did not amplify fecal DNAs from other species. Fecal bacteria endemic to the host species may result from evolution in different types of digestive systems.     

Study of Alu sequences at the hypoxanthine phosphoribosyltransferase (hprt) encoding region of man

The hypoxanthine phosphoribosyltransferase (hprt) encoding region of man is considered rich in Alu sequences; with 49 sequences present within 57 kilobases. Subfamily classification of the Alu sequences and identification of flanking direct repeats has been carried out to detect past rearrangements associated with their insertion into the region. Members of the Alu-J and three Alu-S subfamilies are present, along with the existence of free left arm sequences. Using available data, a comparison is made of the Alu subfamilies present at different gene regions. The heterogeneity in the number of each subfamily present at different genes shows that no one particular subfamily attained saturation in the genome. Several adjacent insertions of Alu sequences are seen at the hprt region. Furthermore two novel sequences are described, there is an incident where one Alu sequence has inserted into the middle poly(A) tract of an existing sequence at the hprt region; while another resulted from an Alu/Alu cross-over event elsewhere in the genome, before insertion into the hprt region. Once inserted, the Alu sequences are rarely subject to loss or rearrangement.     

A ubiquitous family of repeated DNA sequences in the human genome

Renatured DNA from human and many other eukaryotes is known to contain 300-nucleotide duplex regions formed from renatured repeated sequences. These short repeated DNA sequences are widely believed to be interspersed with single copy DNA sequences. In this work we show that at least half of these 300-nucleotide duplexes share a cleavage site for the restriction enzyme AluI. This site is located 170 nucleotides from one end. This Alu family of repeated sequences makes up at least 3% of the genome and is present in several hundred thousand copies.Inverted repeated sequences are also known to contain a short 300-nucleotide duplex region. We find that at least half of the 300-nucleotide duplex regions in inverted repeated sequences also have an AluI restriction site located 170 nucleotides from one end.By driven renaturation techniques, the Alu family is shown to be distributed over a minimum range of 30% to 60% of the genome. (The breadth of this range reflects the presence of inverted repeated sequences which, in part, include the Alu family.) These findings imply that the interspersion pattern of repeated and single copy sequences in human DNA is largely dominated by one family of repeated sequences.     

Conservation of an Intact vif Gene of Human Immunodeficiency Virus Type 1 during Maternal-Fetal Transmission

The human immunodeficiency virus type 1 (HIV-1) vif gene is conserved among most lentiviruses, suggesting that vif is important for natural infection. To determine whether an intact vif gene is positively selected during mother-to-infant transmission, we analyzed vif sequences from five infected mother-infant pairs following perinatal transmission. The coding potential of the vif open reading frame directly derived from uncultured peripheral blood mononuclear cell DNA was maintained in most of the 78,912 bp sequenced. We found that 123 of the 137 clones analyzed showed an 89.8% frequency of intact vif open reading frames. There was a low degree of heterogeneity of vif genes within mothers, within infants, and between epidemiologically linked mother-infant pairs. The distances between vif sequences were greater in epidemiologically unlinked individuals than in epidemiologically linked mother-infant pairs. Furthermore, the epidemiologically linked mother-infant pair vif sequences displayed similar patterns that were not seen in vif sequences from epidemiologically unlinked individuals. The functional domains, including the two cysteines at positions 114 and 133, a serine phosphorylation site at position 144, and the C-terminal basic amino acids essential for vif protein function, were highly conserved in most of the sequences. Phylogenetic analyses of 137 mother-infant pair vif sequences and 187 other available vif sequences from HIV-1 databases revealed distinct clusters for vif sequences from each mother-infant pair and for other vif sequences. Taken together, these findings suggest that vif plays an important role in HIV-1 infection and replication in mothers and their perinatally infected infants.     

Diversity of Babesia bovis merozoite surface antigen genes in the Philippines

Babesia bovis is the causative agent of fatal babesiosis in cattle. In the present study, we investigated the genetic diversity of B. bovis among Philippine cattle, based on the genes that encode merozoite surface antigens (MSAs). Forty-one B. bovis-positive blood DNA samples from cattle were used to amplify the msa-1, msa-2b, and msa-2c genes. In phylogenetic analyses, the msa-1, msa-2b, and msa-2c gene sequences generated from Philippine B. bovis-positive DNA samples were found in six, three, and four different clades, respectively. All of the msa-1 and most of the msa-2b sequences were found in clades that were formed only by Philippine msa sequences in the respective phylograms. While all the msa-1 sequences from the Philippines showed similarity to those formed by Australian msa-1 sequences, the msa-2b sequences showed similarity to either Australian or Mexican msa-2b sequences. In contrast, msa-2c sequences from the Philippines were distributed across all the clades of the phylogram, although one clade was formed exclusively by Philippine msa-2c sequences. Similarities among the deduced amino acid sequences of MSA-1, MSA-2b, and MSA-2c from the Philippines were 62.2–100, 73.1–100, and 67.3–100%, respectively. The present findings demonstrate that B. bovis populations are genetically diverse in the Philippines. This information will provide a good foundation for the future design and implementation of improved immunological preventive methodologies against bovine babesiosis in the Philippines. The study has also generated a set of data that will be useful for futher understanding of the global genetic diversity of this important parasite.     

Phylogenetic Diversity of Sequences of Cyanophage Photosynthetic Gene psbA in Marine and Freshwaters

Many cyanophage isolates which infect the marine cyanobacteria Synechococcus spp. and Prochlorococcus spp. contain a gene homologous to psbA, which codes for the D1 protein involved in photosynthesis. In the present study, cyanophage psbA gene fragments were readily amplified from freshwater and marine samples, confirming their widespread occurrence in aquatic communities. Phylogenetic analyses demonstrated that sequences from freshwaters have an evolutionary history that is distinct from that of their marine counterparts. Similarly, sequences from cyanophages infecting Prochlorococcus and Synechococcus spp. were readily discriminated, as were sequences from podoviruses and myoviruses. Viral psbA sequences from the same geographic origins clustered within different clades. For example, cyanophage psbA sequences from the Arctic Ocean fell within the Synechococcus as well as Prochlorococcus phage groups. Moreover, as psbA sequences are not confined to a single family of phages, they provide an additional genetic marker that can be used to explore the diversity and evolutionary history of cyanophages in aquatic environments.     

Maintenance of an Intact Human Immunodeficiency Virus Type 1 vpr Gene following Mother-to-Infant Transmission

The vpr sequences from six human immunodeficiency virus type 1 (HIV-1)-infected mother-infant pairs following perinatal transmission were analyzed. We found that 153 of the 166 clones analyzed from uncultured peripheral blood mononuclear cell DNA samples showed a 92.17% frequency of intact vpr open reading frames. There was a low degree of heterogeneity of vpr genes within mothers, within infants, and between epidemiologically linked mother-infant pairs. The distances between vpr sequences were greater in epidemiologically unlinked individuals than in epidemiologically linked mother-infant pairs. Moreover, the infants’ sequences displayed patterns similar to those seen in their mothers. The functional domains essential for Vpr activity, including virion incorporation, nuclear import, and cell cycle arrest and differentiation were highly conserved in most of the sequences. Phylogenetic analyses of 166 mother-infant pairs and 195 other available vpr sequences from HIV databases formed distinct clusters for each mother-infant pair and for other vpr sequences and grouped the six mother-infant pairs’ sequences with subtype B sequences. A high degree of conservation of intact and functional vpr supports the notion that vpr plays an important role in HIV-1 infection and replication in mother-infant isolates that are involved in perinatal transmission.     

Genome analysis of ground squirrels of the genus Citellus (Rodentia,Sciuridae) II. DNA sequence organization

The organization of the DNA sequences in five specics of Citellus (C. pygmaeus, C. fulvus, C. major, C. parryi and C. undulatus) was determined from the reassociation kineties of DNA fragments of various lengths and the size distribution of SI-nuclease-resistant duplexes of repetitive DNA. Only 15% of the genome of all the species studied consists of short unique and repeated sequences interspersed with a period less than 2 3 kb, whereas the major part of the genome is occupied by much more extensive sequences of two types, moderately long (3–15 kb) and very long (much more than 15 kb). On the basis of the number of moderately long single-copy sequences the species under study are divided into two groups, coinciding with their division into short-tailed and long-tailed ground squirrels: the short-tailed (C. pygmaeus, C. major and C. fulvus) possess far more such sequences (17–24%) than do the long-tailed ones (C. parryi and C. undulatus) (1–7%). The same division is observed in the amount of very long single-copy sequences. The repeated DNA sequences of Citellus vary widely in size, i.e. from 70 up to some thousands of nucleotide pairs, sequences of more than 1200 nucleotide pairs being most common. In addition, part of the repetitions contain between 70 and 150 base pairs. About one-third of C. parryi repeats (10% of the genome) are characterized by such very short sequences whereas their amont is much less in the other Citellus species (1–4% of the genome).     

Acquisition of new DNA sequences after infection of chicken cells with avian myeloblastosis virus

DNA-RNA hybridization studies between 70S RNA from avian myeloblastosis virus (AMV) and an excess of DNA from (i) AMV-induced leukemic chicken myeloblasts or (ii) a mixture of normal and of congenitally infected K-137 chicken embryos producing avian leukosis viruses revealed the presence of fast- and slow-hybridizing virus-specific DNA sequences. However, the leukemic cells contained twice the level of AMV-specific DNA sequences observed in normal chicken embryonic cells. The fast-reacting sequences were two to three times more numerous in leukemic DNA than in DNA from the mixed embryos. The slow-reacting sequences had a reiteration frequency of approximately 9 and 6, in the two respective systems. Both the fast- and the slow-reacting DNA sequences in leukemic cells exhibited a higher T_m (2 C) than the respective DNA sequences in normal cells. In normal and leukemic cells the slow hybrid sequences appeared to have a T_m which was 2 C higher than that of the fast hybrid sequences. Individual non-virus-producing chicken embryos, either group-specific antigen positive or negative, contained 40 to 100 copies of the fast sequences and 2 to 6 copies of the slowly hybridizing sequences per cell genome. Normal rat cells did not contain DNA that hybridized with AMV RNA, whereas non-virus-producing rat cells transformed by B-77 avian sarcoma virus contained only the slowly reacting sequences. The results demonstrate that leukemic cells transformed by AMV contain new AMV-specific DNA sequences which were not present before infection.     

Bacterial diversity in saline-alkali ponds rearing common carp (Cyprinus carpio) as revealed by 16S rRNA gene sequences

The bacterial diversity in saline-alkali ponds rearing common carp was investigated using the 16S rRNA gene clone library technique. Phylogenetic analysis of the most common and dominant sequences recovered indicated that these sequences fell into the following major lineages, including Proteobacteria (α-, β-, γ-), Actinobacteria, Cyanobacteria, Planctomycetes, Fibrobacteres, Bacteroidetes, Chloroflexi, and unclassified bacteria. Sequence analysis showed that the bacterial diversity was abundant, and the sequences belonging to β-Proteobacteria, α-Proteobacteria and Actinobacteria were predominant. The most sequences in the saline-alkali rearing ponds exhibited low similarity with known bacterial 16S rRNA genes, suggesting that these sequences may represent novel bacteria. In addition, the majority of our sequences were most closely affiliated with sequences retrieved from inland waters of China. These results suggest that the saline-alkali ponds rearing common carp are specific ecologic niches and the distribution of the bacteria may be influenced by geographical factors. This study reports the bacterial diversity in saline-alkali ponds rearing common carp by the culture-independent technique for the first time; therefore, it provides important information for understanding the microbial ecology in saline-alkali rearing ponds and managing the microbial community composition to promote and maintain the health of aquaculture environments.     

Molecular Analysis of the Microbial Diversity Present in the Colonic Wall, Colonic Lumen, and Cecal Lumen of a Pig

Random clones of 16S ribosomal DNA gene sequences were isolated after PCR amplification with eubacterial primers from total genomic DNA recovered from samples of the colonic lumen, colonic wall, and cecal lumen from a pig. Sequences were also obtained for cultures isolated anaerobically from the same colonic-wall sample. Phylogenetic analysis showed that many sequences were related to those of Lactobacillus or Streptococcus spp. or fell into clusters IX, XIVa, and XI of gram-positive bacteria. In addition, 59% of randomly cloned sequences showed less than 95% similarity to database entries or sequences from cultivated organisms. Cultivation bias is also suggested by the fact that the majority of isolates (54%) recovered from the colon wall by culturing were related to Lactobacillus and Streptococcus, whereas this group accounted for only one-third of the sequence variation for the same sample from random cloning. The remaining cultured isolates were mainly Selenomonas related. A higher proportion of Lactobacillus reuteri-related sequences than of Lactobacillus acidophilus- and Lactobacillus amylovorus-related sequences were present in the colonic-wall sample. Since the majority of bacterial ribosomal sequences recovered from the colon wall are less than 95% related to known organisms, the roles of many of the predominant wall-associated bacteria remain to be defined.     

Dockerin-like sequences in cellulases and xylanases from the rumen cellulolytic bacterium Ruminococcus flavefaciens

Recent analysis of the endA cellulase gene from Ruminococcus flavefaciens 17 has revealed that it encodes a product of 759 amino acids that provides the first example of a multidomain cellulase from a Ruminococcus sp. Following the family 5 catalytic domain in the predicted EndA enzyme is a 282 amino acid domain of unknown function for which no close relationship was found to other protein sequences. However, the C-terminal sequences of EndA contain a 34 amino acid threonine-rich linker connected to an 81 amino acid region, both of which show strong similarities to sequences present in two xylanases from R. flavefaciens 17. A distant relationship is evident between regions of the 80 amino acid sequences of EndA, XynD and XynB and the duplicated 23 amino acid dockerin sequences found in cellulolytic Clostridium sp., suggesting that as in Clostridium sp. these sequences could mediate the binding of enzymatic polypeptides to another component in the cell surface enzyme complex of R. flavefaciens.     

Worldwide Distribution of Nitrosococcus oceani, a Marine Ammonia-Oxidizing γ-Proteobacterium, Detected by PCR and Sequencing of 16S rRNA and amoA Genes

Diversity of cultured ammonia-oxidizing bacteria in the γ-subdivision of the Proteobacteria was investigated by using strains isolated from various parts of the world ocean. All the strains were very similar to each other on the basis of the sequences of both the 16S rRNA and ammonia monooxygenase genes and could be characterized as a single species. Sequences were also cloned directly from environmental DNA from coastal Pacific and Atlantic sites, and these sequences represented the first Nitrosococcus oceani-like sequences obtained directly from the ocean. Most of the environmental sequences clustered tightly with those of the cultivated strains, but some sequences could represent new species of Nitrosococcus. These findings imply that organisms similar to the cultivated N. oceani strains have a worldwide distribution.