A mutation in Flavobacterium psychrophilum tlpB inhibits gliding motility and induces biofilm formation

Flavobacterium psychrophilum is a psychrotrophic, fish-pathogenic bacterium belonging to the Cytophaga-Flavobacterium-Bacteroides group. Tn4351-induced mutants deficient in gliding motility, growth on iron-depleted media, and extracellular proteolytic activity were isolated. Some of these mutants were affected in only one of these characteristics, whereas others had defects in two or more. FP523, a mutant deficient in all of these properties, was studied further. FP523 had a Tn4351 insertion in tlpB (thiol oxidoreductase-like protein gene), which encodes a 41.4-kDa protein whose sequence does not exhibit high levels of similar to the sequences of proteins having known functions. TlpB has two domains; the N-terminal domains has five transmembrane regions, whereas the C-terminal domains has the Cys-X-X-Cys motif and other conserved motifs characteristic of thiol:disulfide oxidoreductases. Quantitative analysis of the thiol groups of periplasmic proteins revealed that TlpB is required for reduction of these groups. The tlpB gene is part of the fpt (F. psychrophilum thiol oxidoreductase) operon that contains two other genes, tlpA and tpiA, which encode a thiol:disulfide oxidoreductase and a triosephosphate isomerase, respectively. FP523 exhibited enhanced biofilm formation and decreased virulence and cytotoxicity. Complementation with the tlpB loci restored the wild-type phenotype. Gliding motility and biofilm formation appear to be antagonistic properties, which are both affected by TlpB.     

Identification of genes that are associated with DNA repeats in prokaryotes

Using in silico analysis we studied a novel family of repetitive DNA sequences that is present among both domains of the prokaryotes (Archaea and Bacteria), but absent from eukaryotes or viruses. This family is characterized by direct repeats, varying in size from 21 to 37 bp, interspaced by similarly sized non-repetitive sequences. To appreciate their characteri-stic structure, we will refer to this family as the clustered regularly interspaced short palindromic repeats (CRISPR). In most species with two or more CRISPR loci, these loci were flanked on one side by a common leader sequence of 300-500 b. The direct repeats and the leader sequences were conserved within a species, but dissimilar between species. The presence of multiple chromosomal CRISPR loci suggests that CRISPRs are mobile elements. Four CRISPR-associated (cas) genes were identified in CRISPR-containing prokaryotes that were absent from CRISPR-negative prokaryotes. The cas genes were invariably located adjacent to a CRISPR locus, indicating that the cas genes and CRISPR loci have a functional relationship. The cas3 gene showed motifs characteristic for helicases of the superfamily 2, and the cas4 gene showed motifs of the RecB family of exonucleases, suggesting that these genes are involved in DNA metabolism or gene expression. The spatial coherence of CRISPR and cas genes may stimulate new research on the genesis and biological role of these repeats and genes.     

A Mutation in Flavobacterium psychrophilum tlpB Inhibits Gliding Motility and Induces Biofilm Formation

Flavobacterium psychrophilum is a psychrotrophic, fish-pathogenic bacterium belonging to the Cytophaga-Flavobacterium-Bacteroides group. Tn4351-induced mutants deficient in gliding motility, growth on iron-depleted media, and extracellular proteolytic activity were isolated. Some of these mutants were affected in only one of these characteristics, whereas others had defects in two or more. FP523, a mutant deficient in all of these properties, was studied further. FP523 had a Tn4351 insertion in tlpB (thiol oxidoreductase-like protein gene), which encodes a 41.4-kDa protein whose sequence does not exhibit high levels of similar to the sequences of proteins having known functions. TlpB has two domains; the N-terminal domains has five transmembrane regions, whereas the C-terminal domains has the Cys-X-X-Cys motif and other conserved motifs characteristic of thiol:disulfide oxidoreductases. Quantitative analysis of the thiol groups of periplasmic proteins revealed that TlpB is required for reduction of these groups. The tlpB gene is part of the fpt (F. psychrophilum thiol oxidoreductase) operon that contains two other genes, tlpA and tpiA, which encode a thiol:disulfide oxidoreductase and a triosephosphate isomerase, respectively. FP523 exhibited enhanced biofilm formation and decreased virulence and cytotoxicity. Complementation with the tlpB loci restored the wild-type phenotype. Gliding motility and biofilm formation appear to be antagonistic properties, which are both affected by TlpB.     

Patterns of molecular evolution among paralogous floral homeotic genes.

The plant MADS-box regulatory gene family includes several loci that control different aspects of inflorescence and floral development. Orthologs to the Arabidopsis thaliana MADS-box floral meristem genes APETALA1 and CAULIFLOWER and the floral organ identity genes APETALA3 and PISTILLATA were isolated from the congeneric species Arabidopsis lyrata. Analysis of these loci between these two Arabidopsis species, as well as three other more distantly related taxa, reveal contrasting dynamics of molecular evolution between these paralogous floral regulatory genes. Among the four loci, the CAL locus evolves at a significantly faster rate, which may be associated with the evolution of genetic redundancy between CAL and AP1. Moreover, there are significant differences in the distribution of replacement and synonymous substitutions between the functional gene domains of different floral homeotic loci. These results indicate that divergence in developmental function among paralogous members of regulatory gene families is accompanied by changes in rate and pattern of sequence evolution among loci.     

A gain-of-function screen for genes that affect the development of the Drosophila adult external sensory organ

The Drosophila adult external sensory organ, comprising a neuron and its support cells, is derived from a single precursor cell via several asymmetric cell divisions. To identify molecules involved in sensory organ development, we conducted a tissue-specific gain-of-function screen. We screened 2293 independent P-element lines established by P. Rorth and identified 105 lines, carrying insertions at 78 distinct loci, that produced misexpression phenotypes with changes in number, fate, or morphology of cells of the adult external sensory organ. On the basis of the gain-of-function phenotypes of both internal and external support cells, we subdivided the candidate lines into three classes. The first class (52 lines, 40 loci) exhibits partial or complete loss of adult external sensory organs. The second class (38 lines, 28 loci) is associated with increased numbers of entire adult external sensory organs or subsets of sensory organ cells. The third class (15 lines, 10 loci) results in potential cell fate transformations. Genetic and molecular characterization of these candidate lines reveals that some loci identified in this screen correspond to genes known to function in the formation of the peripheral nervous system, such as big brain, extra macrochaetae, and numb. Also emerging from the screen are a large group of previously uncharacterized genes and several known genes that have not yet been implicated in the development of the peripheral nervous system.     

Expression and genome organization of resistance gene analogs in soybean.

Sequence analysis of cloned plant disease-resistance genes reveals a number of conserved domains. Researchers have used these domains to amplify analogous sequences, resistance gene analogs (RGAs), from soybean and other crops. Many of these RGAs map in close proximity to known resistance genes. While this technique is useful in identifying potential disease resistance loci, identifying the functional resistance gene from a cluster of homologs requires sequence information from outside of these conserved domains. To study RGA expression and to determine the extent of their similarity to other plant resistance genes, two soybean cDNA libraries (root and epicotyl) were screened by hybridization with RGA class-specific probes. cDNAs hybridizing to RGA probes were detected in each library. Two types of cDNAs were identified. One type was full-length and contained several disease-resistance gene (R-gene) signatures. The other type contained several deletions within these signatures. Sequence analyses of the cDNA clones placed them in the Toll-Interleukin-1 receptor, nucleotide binding domain, and leucine-rich repeat family of disease-resistance genes. Using clone-specific primers from within the 3' end of the LRRs, we were able to map two cDNA clones (LM6 and MG13) to a BAC contig that is known to span a cluster of disease-resistance genes.     

The Pax-homeobox gene eyegone is involved in the subdivision of the thorax of Drosophila

The eyegone (eyg) gene is known to be involved in the development of the eye structures of Drosophila. We show that eyg and its related gene, twin of eyegone (toe), are also expressed in part of the anterior compartment of the adult mesothorax (notum). We report experiments concerning the role of these genes in the notum. In the absence of eyg function the anterior-central region does not develop, whereas ectopic activity of either eyg or toe induces the formation of the anterior-central pattern in the posterior or lateral region of the notum. These results demonstrate that eyg and toe play a role in the genetic subdivision of the notum, although the experiments indicate that eyg exerts the principal function. However, by itself the Eyg product cannot induce the formation of notum patterns; its thoracic function requires co-expression with the Iroquois (Iro) genes. We show that the restriction of eyg activity to the anterior-central region of the wing disc is achieved by the antagonistic regulatory activities of the Iro and pnr genes, which promote eyg expression, and those of the Hh and Dpp pathways, which act as repressors. We argue that eyg is a subordinate gene of the Iro genes, and that pnr mediates their thoracic patterning function. The activity of eyg gives rise to a new notum subdivision that acts upon the pre-extant one generated by the Iro genes and pnr. As a result the notum becomes subdivided into four distinct genetic domains.     

A Taxonomy of Bacterial Microcompartment Loci Constructed by a Novel Scoring Method

Bacterial microcompartments (BMCs) are proteinaceous organelles involved in both autotrophic and heterotrophic metabolism. All BMCs share homologous shell proteins but differ in their complement of enzymes; these are typically encoded adjacent to shell protein genes in genetic loci, or operons. To enable the identification and prediction of functional (sub)types of BMCs, we developed LoClass, an algorithm that finds putative BMC loci and inventories, weights, and compares their constituent pfam domains to construct a locus similarity network and predict locus (sub)types. In addition to using LoClass to analyze sequences in the Non-redundant Protein Database, we compared predicted BMC loci found in seven candidate bacterial phyla (six from single-cell genomic studies) to the LoClass taxonomy. Together, these analyses resulted in the identification of 23 different types of BMCs encoded in 30 distinct locus (sub)types found in 23 bacterial phyla. These include the two carboxysome types and a divergent set of metabolosomes, BMCs that share a common catalytic core and process distinct substrates via specific signature enzymes. Furthermore, many Candidate BMCs were found that lack one or more core metabolosome components, including one that is predicted to represent an entirely new paradigm for BMC-associated metabolism, joining the carboxysome and metabolosome. By placing these results in a phylogenetic context, we provide a framework for understanding the horizontal transfer of these loci, a starting point for studies aimed at understanding the evolution of BMCs. This comprehensive taxonomy of BMC loci, based on their constituent protein domains, foregrounds the functional diversity of BMCs and provides a reference for interpreting the role of BMC gene clusters encoded in isolate, single cell, and metagenomic data. Many loci encode ancillary functions such as transporters or genes for cofactor assembly; this expanded vocabulary of BMC-related functions should be useful for design of genetic modules for introducing BMCs in bioengineering applications.     

Biosynthesis of phytohormones in algae

Like in the case of higher plants, algal growth and development are controlled by the hormonal regulatory system. Essentially all known phytohormones were identified in various algal taxa, and the range of their physiological activities was confirmed. At the same time, our knowledge of enzymes involved in the phytohormone synthesis in algae is rather limited. Data concerning genes encoding these enzymes are still more fragmentary. Current data about proteomes of some algae allow the revealing of amino acid sequences with homology to those of the higher plant enzymes and their conserved domains.     

Genetic interactions between clock mutations in Neurospora crassa: can they help us to understand complexity?

Recent work on circadian clocks in Neurospora has primarily focused on the frequency (frq) and white-collar (wc) loci. However, a number of other genes are known that affect either the period or temperature compensation of the rhythm. These include the period (no relationship to the period gene of Drosophila) genes and a number of genes that affect cellular metabolism. How these other loci fit into the circadian system is not known, and metabolic effects on the clock are typically not considered in single-oscillator models. Recent evidence has pointed to multiple oscillators in Neurospora, at least one of which is predicted to incorporate metabolic processes. Here, the Neurospora clock-affecting mutations will be reviewed and their genetic interactions discussed in the context of a more complex clock model involving two coupled oscillators: a FRQ/WC-based oscillator and a 'frq-less' oscillator that may involve metabolic components.     

High-resolution copy-number variation map reflects human olfactory receptor diversity and evolution

Olfactory receptors (ORs), which are involved in odorant recognition, form the largest mammalian protein superfamily. The genomic content of OR genes is considerably reduced in humans, as reflected by the relatively small repertoire size and the high fraction ( approximately 55%) of human pseudogenes. Since several recent low-resolution surveys suggested that OR genomic loci are frequently affected by copy-number variants (CNVs), we hypothesized that CNVs may play an important role in the evolution of the human olfactory repertoire. We used high-resolution oligonucleotide tiling microarrays to detect CNVs across 851 OR gene and pseudogene loci. Examining genomic DNA from 25 individuals with ancestry from three populations, we identified 93 OR gene loci and 151 pseudogene loci affected by CNVs, generating a mosaic of OR dosages across persons. Our data suggest that approximately 50% of the CNVs involve more than one OR, with the largest CNV spanning 11 loci. In contrast to earlier reports, we observe that CNVs are more frequent among OR pseudogenes than among intact genes, presumably due to both selective constraints and CNV formation biases. Furthermore, our results show an enrichment of CNVs among ORs with a close human paralog or lacking a one-to-one ortholog in chimpanzee. Interestingly, among the latter we observed an enrichment in CNV losses over gains, a finding potentially related to the known diminution of the human OR repertoire. Quantitative PCR experiments performed for 122 sampled ORs agreed well with the microarray results and uncovered 23 additional CNVs. Importantly, these experiments allowed us to uncover nine common deletion alleles that affect 15 OR genes and five pseudogenes. Comparison to the chimpanzee reference genome revealed that all of the deletion alleles are human derived, therefore indicating a profound effect of human-specific deletions on the individual OR gene content. Furthermore, these deletion alleles may be used in future genetic association studies of olfactory inter-individual differences.     

Evolution of EF-hand calcium-modulated proteins. V. The genes encoding EF-hand proteins are not clustered in mammalian genomes

Summary The chromosomal assignments of genes belonging to the EF-hand family which have a common origin are compiled in this article. So far data are available from 27 human gene loci belonging to 6 subfamilies and 8 murine loci belonging to 4 subfamilies. Chromosomal localization has been obtained by somatic-cell hybrid analysis using the Southern blot technique or PCR amplification, metaphase spread in situ hybridization, or isolation of the particular genes from chromosome-specific libraries. Except for genes of the S-100 alpha proteins which are grouped on human chromosome 1q12-25 and mouse chromosome 3, no linkage has been found for genes encoding EF-hand proteins, indicating absence of selective pressure for maintaining chromosomal clustering. Six of these genes map to known syntenic groups conserved in the human and mouse genomes. This suggests that chromosomal translocations occurred before divergence of these species. The possible significance of chromosomal positioning with respect to nearby located known genes and genetic disease loci is discussed.     

Identification and mapping on human chromosome 19 of chromosome-specific repeated element preferentially binding with nuclear matrix]

From a library of sequences binding preferentially to nuclear matrix (matrix attachment regions, MARs), a fragment of about 300 bp in length (CEA (carcinoembryonic antigen)-MAR) was isolated and characterized. The CEA-MAR sequence was found in more than ten loci of chromosome 19 containing elements similar to genes of the CEA family. No sequences of this group were found on other human chromosomes. Two CEA-MAR-containing loci were sequenced, and sequences for another seven loci were found in GenBank. A comparative analysis of CEA-MARs and the flanking sequences is reported. Based on the sequence of the CEA-containing chromosome 19 loci, a hypothetical model of the domain structure of a 2-Mb chromosome region was constructed and the mutual arrangement of CEA-MARs and genes of CEA family was elucidated. The CEA-MARs were located 5-20 kb downstream of the CEA genes. These results suggest that the duplication unit of the CEA family may coincide with chromatin domains containing these genes.     

A Screen for Genetic Loci Required for Hypodermal Cell and Glial-like Cell Development during Caenorhabditis Elegans Embryogenesis

The Caenorhabditis elegans lin-26 gene is expressed in all nonneuronal ectodermal cells. To identify genes required to specify the fates of ectodermal cells, we have conducted screens designed to identify loci whose zygotic function would be required for normal lin-26 expression. First, we examined 90 deficiencies covering 75% of the genome; second, we examined the progeny of 3600 genomes after EMS mutagenesis. We identified six loci that appear to be required for normal lin-26 expression. We argue that the deficiency eDf19 deletes a gene involved in specifying hypodermal cell fates. The genes emb-29 (previously known) and ale-1 (newly found) could be involved in a cell cycle function and/or in specifying the fates of some precursors within different lineages that generate hypodermal cells and nonectodermal cells. We argue that the overlapping deficiencies qDf7, qDf8 and qDf9 delete a gene required to limit the number of nonneuronal ectodermal cells. We suggest that the deficiencies ozDf2, itDf2 and nDf42 delete genes required, directly or indirectly, to repress lin-26 expression in cells that normally do not express lin-26. We discuss the implications of these findings concerning the generation of the ectoderm.     

Genomic and population analyses of the mating type loci in Coccidioides species reveal evidence for sexual reproduction and gene acquisition

Coccidioides species, the fungi responsible for the valley fever disease, are known to reproduce asexually through the production of arthroconidia that are the infectious propagules. The possible role of sexual reproduction in the survival and dispersal of these pathogens is unexplored. To determine the potential for mating of Coccidioides, we analyzed genome sequences and identified mating type loci characteristic of heterothallic ascomycetes. Coccidioides strains contain either a MAT1-1 or a MAT1-2 idiomorph, which is 8.1 or 9 kb in length, respectively, the longest reported for any ascomycete species. These idiomorphs contain four or five genes, respectively, more than are present in the MAT loci of most ascomycetes. Along with their cDNA structures, we determined that all genes in the MAT loci are transcribed. Two genes frequently found in common sequences flanking MAT idiomorphs, APN2 and COX13, are within the MAT loci in Coccidioides, but the MAT1-1 and MAT1-2 copies have diverged dramatically from each other. Data indicate that the acquisition of these genes in the MAT loci occurred prior to the separation of Coccidioides from Uncinocarpus reesii. An analysis of 436 Coccidioides isolates from patients and the environment indicates that in both Coccidioides immitis and C. posadasii, there is a 1:1 distribution of MAT loci, as would be expected for sexually reproducing species. In addition, an analysis of isolates obtained from 11 soil samples demonstrated that at three sampling sites, strains of both mating types were present, indicating that compatible strains were in close proximity in the environment.     

Patterning of proneuronal and inter-proneuronal domains by hairy- and enhancer of split-related genes in zebrafish neuroectoderm

In teleosts and amphibians, the proneuronal domains, which give rise to primary-motor, primary-inter and Rohon-Beard (RB) neurons, are established at the beginning of neurogenesis as three longitudinal stripes along the anteroposterior axis in the dorsal ectoderm. The proneuronal domains are prefigured by the expression of basic helix-loop-helix (bHLH) proneural genes, and separated by domains (inter-proneuronal domains) that do not express the proneural genes. Little is known about how the formation of these domains is spatially regulated. We have found that the zebrafish hairy- and enhancer of split-related (Her) genes her3 and her9 are expressed in the inter-proneuronal domains, and are required for their formation. her3 and her9 expression was not regulated by Notch signaling, but rather controlled by positional cues, in which Bmp signaling is involved. Inhibition of Her3 or Her9 by antisense morpholino oligonucleotides led to ectopic expression of the proneural genes in part of the inter-proneuronal domains. Combined inhibition of Her3 and Her9 induced ubiquitous expression of proneural and neuronal genes in the neural plate, and abolished the formation of the inter-proneuronal domains. Furthermore, inhibition of Her3/Her9 and Notch signaling led to ubiquitous and homogeneous expression of proneural and neuronal genes in the neural plate, revealing that Her3/Her9 and Notch signaling have distinct roles in neurogenesis. These data indicate that her3 and her9 function as prepattern genes that link the positional dorsoventral polarity information in the posterior neuroectoderm to the spatial regulation of neurogenesis.