Genes Encoding Membrane Proteins Research Articles

Gene expression patterns in larval Schistosoma mansoni associated with infection of the mammalian host.

BackgroundThe infective schistosome cercaria develops within the intramolluscan daughter sporocyst from an undifferentiated germ ball, during which synthesis of proteins essential for infection occurs. When the aquatic cercaria locates the mammalian host it rapidly penetrates into the epidermis using glandular secretions. It then undergoes metamorphosis into the schistosomulum, including replacement of its tegument surface membranes, a process taking several days before it exits the skin. Patterns of gene expression underlying this transition have been characterised.Methods and Principal FindingsAll gene models from the S. mansoni genome (www.GeneDB.org) were incorporated into a high-density oligonucleotide array. Double-stranded cDNA from germ balls, cercariae, and day 3 schistosomula was hybridised to the array without amplification. Statistical analysis was performed using Bioconductor to reveal differentially transcribed loci. Genes were categorised on the basis of biological process, tissue association or molecular function to aid understanding of the complex processes occurring. Genes necessary for DNA replication were enriched only in the germ ball, while those involved in translation were up-regulated in the germ ball and/or day 3 schistosomulum. Different sets of developmental genes were up-regulated at each stage. A large number of genes encoding elastases and invadolysins, and some venom allergen-like proteins were up-regulated in the germ ball, those encoding cysteine and aspartic proteases in the cercaria and schistosomulum. Micro exon genes encoding variant secreted proteins were highly up-regulated in the schistosomulum along with tegument and gut-associated genes, coincident with remodelling of the parasite body. Genes encoding membrane proteins were prominently up-regulated in the cercaria and/or day 3 schistosomulum.Conclusions/SignificanceOur study highlights an expanded number of transcripts encoding proteins potentially involved in skin invasion. It illuminates the process of metamorphosis into the schistosomulum and highlights the very early activation of gut-associated genes whilst revealing little change in the parasite's energy metabolism or stress responses.

Differences in dinucleotide frequencies of thermophilic genes encoding water soluble and membrane proteins

The occurrence frequencies of the dinucleotides of genes of three thermophilic and three mesophilic species from both archaea and eubacteria were investigated in this study. The genes encoding water soluble proteins were rich in the dinucleotides of purine dimers, whereas the genes encoding membrane proteins were rich in pyrimidine dimers. The dinucleotides of purine dimers are the counterparts of pyrimidine dimers in a double-stranded DNA. The purine/pyrimidine dimers were favored in the thermophiles but not in the mesophiles, based on comparisons of observed and expected frequencies. This finding is in agreement with our previous study which showed that purine/pyrimidine dimers are positive factors that increase the thermal stability of DNA. The dinucleotides AA, AG, and GA are components of the codons of charged residues of Glu, Asp, Lys, and Arg, and the dinucleotides TT, CT, and TC are components of the codons of hydrophobic residues of Leu, Ile, and Phe. This is consistent with the suitabilities of the different amino acid residues for water soluble and membrane proteins. Our analysis provides a picture of how thermophilic species produce water soluble and membrane proteins with distinctive characters: the genes encoding water soluble proteins use DNA sequences rich in purine dimers, and the genes encoding membrane proteins use DNA sequences rich in pyrimidine dimers on the opposite strand.

Genetic variability of Chlamydophila abortus strains assessed by PCR-RFLP analysis of polymorphic membrane protein-encoding genes

This study used PCR-RFLP to investigate the genetic variability of pmp-encoding genes from fifty-two Chlamydophila abortus ( C. abortus) strains originating from abortion cases from various geographical regions and host species. Six primer pairs were used to PCR-amplify DNA fragments encoding eighteen pmps. PCR products were digested using four restriction endonucleases and Bayesian methodologies were used to compare RFLP profiles and assign strains to a RFLP genotype. Strains could be assigned to 2 genotypes in the region encoding pmp18D, 3 genotypes in the regions encoding pmp1A-pmp2B, pmp3E-pmp6H and pmp11G-pmp15G, 4 genotypes in the region encoding pmp7G-pmp10G and 5 genotypes in the region encoding pmp16G- pmp17G. In all regions, the majority of strains (88.4–96.1%) had the same genotype as the reference strain S26/3. No correlation could be made between genotype, host species or geographical origin except for the two variant Greek strains, LLG and POS, which formed a discrete genotype in all pmp-encoding regions except pmp18D. Relative rates of evolution calculated for each pmp-encoding gene locus suggest that differing selective pressures and functional constraints may exist on C. abortus polymorphic membrane proteins. These findings suggest that although intraspecies heterogeneity of pmp-encoding genes in C. abortus is low, the sequence heterogeneity should be an important consideration when using pmps as the basis for novel diagnostics or vaccine development.

Multiple Infection of Apple Trees by Distinct Strains of ‘Candidatus Phytoplasma mali’ and Its Pathological Relevance

Forty-eight apple trees infected by 'Candidatus Phytoplasma mali' were examined using single-strand conformation polymorphism (SSCP) and sequence analysis of a variable hflB gene fragment and the immunodominant membrane protein-encoding imp gene. SSCP analysis of polymerase chain reaction-amplified hflB gene fragments revealed diverse profiles, differing in number and position of the bands. The 'Ca. P. mali' content of a single infected apple tree was termed an accession. Cloning of fragments from accessions that yielded fewer bands resulted in clone populations showing uniform or moderately polymorphic SSCP patterns and largely homogenous nucleotide sequences. In contrast, inserts from accessions yielding more bands were heterogeneous and formed two to four distinct groups of profiles. DNA fragments from such accessions were diverse and clustered distantly when subjected to phylogenetic analysis, mostly as two homogenous groups plus one or a few other sequences. Similar results were obtained upon imp gene examination. The collective data indicate that accessions exhibiting more complex patterns were composed of two or three distinct 'Ca. P. mali' strains. There is evidence that multiple infections are of pathological relevance due to strain interactions leading to shifts in the populations. In triply infected trees of one accession, no specific symptoms were induced by the presence of two of the strains. The rare appearance of pronounced symptoms was associated with a separate strain that possessed a unique SSCP profile and a unique hflB sequence. The two mild strains from this apple accession also induced only mild symptoms on periwinkle and tobacco and occurred specifically in one of these plants.

Quantitative Proteome and Transcriptome Analysis of the Archaeon Thermoplasma acidophilum Cultured under Aerobic and Anaerobic Conditions

A comparative proteome and transcriptome analysis of Thermoplasma acidophilum cultured under aerobic and anaerobic conditions has been performed. One-thousand twenty-five proteins were identified covering 88% of the cytosolic proteome. Using a label-free quantitation method, we found that approximately one-quarter of the identified proteome (263 proteins) were significantly induced (>2 fold) under anaerobic conditions. Thirty-nine macromolecular complexes were identified, of which 28 were quantified and 15 were regulated under anaerobiosis. In parallel, a whole genome cDNA microarray analysis was performed showing that the expression levels of 445 genes were influenced by the absence of oxygen. Interestingly, more than 40% of the membrane protein-encoding genes (145 out of 335 ORFs) were up- or down-regulated at the mRNA level. Many of these proteins are functionally associated with extracellular protein or peptide degradation or ion and amino acid transport. Comparison of the transcriptome and proteome showed only a weak positive correlation between mRNA and protein expression changes, which is indicative of extensive post-transcriptional regulatory mechanisms in T. acidophilum. Integration of transcriptomics and proteomics data generated hypotheses for physiological adaptations of the cells to anaerobiosis, and the quantitative proteomics data together with quantitative analysis of protein complexes provide a platform for correlation of MS-based proteomics studies with cryo-electron tomography-based visual proteomics approaches.

A Mycobacterium tuberculosis Sigma Factor Network Responds to Cell-Envelope Damage by the Promising Anti-Mycobacterial Thioridazine

BackgroundNovel therapeutics are urgently needed to control tuberculosis (TB). Thioridazine (THZ) is a candidate for the therapy of multidrug and extensively drug-resistant TB.Methodology/Principal FindingsWe studied the impact of THZ on Mycobacterium tuberculosis (Mtb) by analyzing gene expression profiles after treatment at the minimal inhibitory (1x MIC) or highly inhibitory (4x MIC) concentrations between 1–6 hours. THZ modulated the expression of genes encoding membrane proteins, efflux pumps, oxido-reductases and enzymes involved in fatty acid metabolism and aerobic respiration. The Rv3160c-Rv3161c operon, a multi-drug transporter and the Rv3614c/3615c/3616c regulon, were highly induced in response to THZ. A significantly high number of Mtb genes co-expressed with σB (the σB regulon) was turned on by THZ treatment. σB has recently been shown to protect Mtb from envelope-damage. We hypothesized that THZ damages the Mtb cell-envelope, turning on the expression of the σB regulon. Consistent with this hypothesis, we present electron-microscopy data which shows that THZ modulates cell-envelope integrity. Moreover, the Mtb mutants in σH and σE, two alternate stress response sigma factors that induce the expression of σB, exhibited higher sensitivity to THZ, indicating that the presence and expression of σB allows Mtb to resist the impact of THZ. Conditional induction of σB levels increased the survival of Mtb in the presence of THZ.Conclusions/SignificanceTHZ targets different pathways and can thus be used as a multi-target inhibitor itself as well as provide strategies for multi-target drug development for combination chemotherapy. Our results show that the Mtb sigma factor network comprising of σH, σE and σB plays a crucial role in protecting the pathogen against cell-envelope damage.

Phylogenetic Analysis of the MS4A and TMEM176 Gene Families

BackgroundThe MS4A gene family in humans includes CD20 (MS4A1), FcRβ (MS4A2), Htm4 (MS4A3), and at least 13 other syntenic genes encoding membrane proteins, most having characteristic tetraspanning topology. Expression of MS4A genes is variable in tissues throughout the body; however, several are limited to cells in the hematopoietic system where they have known roles in immune cell functions. Genes in the small TMEM176 group share significant sequence similarity with MS4A genes and there is evidence of immune function of at least one of the encoded proteins. In this study, we examined the evolutionary history of the MS4A/TMEM176 families as well as tissue expression of the phylogenetically earliest members, in order to investigate their possible origins in immune cells.Principal FindingsOrthologs of human MS4A genes were found only in mammals; however, MS4A gene homologs were found in most jawed vertebrates. TMEM176 genes were found only in mammals and bony fish. Several unusual MS4A genes having 2 or more tandem MS4A sequences were identified in the chicken (Gallus gallus) and early mammals (opossum, Monodelphis domestica and platypus, Ornithorhyncus anatinus). A large number of highly conserved MS4A and TMEM176 genes was found in zebrafish (Danio rerio). The most primitive organism identified to have MS4A genes was spiny dogfish (Squalus acanthus). Tissue expression of MS4A genes in S. acanthias and D. rerio showed no evidence of expression restricted to the hematopoietic system.Conclusions/SignificanceOur findings suggest that MS4A genes first appeared in cartilaginous fish with expression outside of the immune system, and have since diversified in many species into their modern forms with expression and function in both immune and nonimmune cells.

Gp93, the Drosophila GRP94 ortholog, is required for gut epithelial homeostasis and nutrient assimilation-coupled growth control

GRP94, the endoplasmic reticulum Hsp90, is a metazoan-restricted chaperone essential for early development in mammals, yet dispensable for mammalian cell viability. This dichotomy suggests that GRP94 is required for the functional expression of secretory and/or membrane proteins that enable the integration of cells into tissues. To explore this hypothesis, we have identified the Drosophila ortholog of GRP94, Gp93, and report that Gp93 is an essential gene in Drosophila. Loss of zygotic Gp93 expression is late larval-lethal and causes prominent defects in the larval midgut, the sole endoderm-derived larval tissue. Gp93 mutant larvae display pronounced defects in the midgut epithelium, with aberrant copper cell structure, markedly reduced gut acidification, atypical septate junction structure, depressed gut motility, and deficits in intestinal nutrient uptake. The metabolic consequences of the loss of Gp93-expression are profound; Gp93 mutant larvae exhibit a starvation-like metabolic phenotype, including suppression of insulin signaling and extensive mobilization of amino acids and triglycerides. The defects in copper cell structure/function accompanying loss of Gp93 expression resemble those reported for mutations in labial, an endodermal homeotic gene required for copper cell specification, and α-spectrin, thus suggesting an essential role for Gp93 in the functional expression of secretory/integral membrane protein-encoding lab protein target genes and/or integral membrane protein(s) that interact with the spectrin cytoskeleton to confer epithelial membrane specialization.

Small variable segments constitute a major type of diversity of bacterial genomes at the species level

Comparison of all available genomes of three bacterial species suggests that a large part of genome diversity is contributed by short regions.

CmdABCDEF, a cluster of genes encoding membrane proteins for differentiation and antibiotic production in Streptomyces coelicolorA3(2)

BackgroundStreptomyces coelicolor is the most studied Streptomyces species and an excellent model for studying differentiation and antibiotic production. To date, many genes have been identified to be required for its differentiation (e.g. bld genes for aerial growth and whi genes for sporulation) and antibiotics production (including actII-orf4, redD, cdaR as pathway-specific regulatory genes and afsR, absA1/A2 as pleiotropic regulatory genes).ResultsA gene cluster containing six genes (SCO4126-4131) was proved to be co-transcribed in S. coelicolor. Deletions of cmdABCDEF (SCO4126-4131) displayed defective sporulation including formation of aberrant branches, and abnormalities in chromosome segregation and spore septation. Disruption mutants of apparently orthologous genes of S. lividans and S. avermitilis also showed defective sporulation, implying that the role of these genes is similar among Streptomyces. Transcription of cmdB, and therefore presumably of the whole operon, was regulated developmentally. Five of the encoded proteins (CmdA, C, D, E, F) were predicted membrane proteins. The other, CmdB, a predicted ATP/GTP-binding protein with an ABC-transporter-ATPase domain shown here to be essential for its function, was also located on the cell membrane. These results indicate that CmdABCDEF proteins mainly affect Streptomyces differentiation at an early stage of aerial hyphae formation, and suggest that these proteins may form a complex on cell membrane for proper segregation of chromosomes. In addition, deletions of cmdABCDEF also revealed over-production of blue-pigmented actinorhodin (Act) via activation of transcription of the pathway-specific regulatory gene actII-orf4 of actinorhodin biosynthesis.ConclusionIn this study, six co-transcribed genes cmdABCDEF were identified by their effects on differentiation and antibiotic production in Streptomyces coelicolor A3(2). These six membrane-located proteins are possibly assembled into a complex to function.

Endometrial biopsy-induced gene modulation: first evidence for the expression of bladder-transmembranal uroplakin Ib in human endometrium

To explore the possibility that endometrial injury modulates the expression of specific genes that may increase uterine receptivity. Controlled clinical study. Clinical IVF unit and academic research center. IVF patients with 28- to 30-day menstrual cycles. Endometrial biopsies from two groups of patients were collected on days 20-21 of their spontaneous menstrual cycle. The experimental, but not the control, group underwent biopsies on days 11-13 and 21-24 of their preceding cycle. Global endometrial gene expression and specific analysis of uroplakin Ib (UPIb) mRNA level throughout the menstrual cycle. Local injury modulated the expression of a wide variety of genes. One of the prominently up-regulated genes was the bladder transmembranal protein, UPIb, whose expression by the endometrium is shown here for the first time. Endometrial UPIb mRNA increases after biopsy in the same cycle wct 2with an additional elevation in the following cycle. Immunohistochemical analysis localized the UPIb protein to the glandular-epithelial cells. Genes encoding other membrane proteins such as adipose differentiation-related protein and mucin 1, transmembrane, were also up-regulated. The biopsy-induced increase in the expression of UPIb and other genes encoding membrane proteins supports the possible importance of the membrane structure and stability during implantation. The specific role of UPIb in uterine receptivity should be elucidated.

Congenital Chloride-losing Diarrhea Causing Mutations in the STAS Domain Result in Misfolding and Mistrafficking of SLC26A3

Congenital chloride-losing diarrhea (CLD) is a genetic disorder causing watery stool and dehydration. Mutations in SLC26A3 (solute carrier 26 family member 3), which functions as a coupled Cl(-)/HCO(3)(-) exchanger, cause CLD. SLC26A3 is a membrane protein predicted to contain 12 transmembrane-spanning alpha-helices and a C-terminal STAS (sulfate transporters and anti-sigma-factor) domain homologous to the bacterial anti-sigma-factor antagonists. The STAS domain is required for SLC26A3 Cl(-)/HCO(3)(-) exchange function and for the activation of cystic fibrosis transmembrane conductance regulator by SLC26A3. Here we investigate the molecular mechanism(s) by which four CLD-causing mutations (DeltaY526/7, I544N, I675/6ins, and G702Tins) in the STAS domain lead to disease. In a heterologous mammalian expression system biochemical, immunohistochemical, and ion transport experiments suggest that the four CLD mutations cause SLC26A3 transporter misfolding and/or mistrafficking. Expression studies with the isolated STAS domain suggest that the I675/6ins and G702Tins mutations disrupt the STAS domain directly, whereas limited proteolysis experiments suggest that the DeltaY526/7 and I544N mutations affect a later step in the folding and/or trafficking pathway. The data suggest that these CLD-causing mutations cause disease by at least two distinct molecular mechanisms, both ultimately leading to loss of functional protein at the plasma membrane.

SLAC1 is required for plant guard cell S-type anion channel function in stomatal signalling.

Stomatal pores, formed by two surrounding guard cells in the epidermis of plant leaves, allow influx of atmospheric carbon dioxide in exchange for transpirational water loss. Stomata also restrict the entry of ozone--an important air pollutant that has an increasingly negative impact on crop yields, and thus global carbon fixation and climate change. The aperture of stomatal pores is regulated by the transport of osmotically active ions and metabolites across guard cell membranes. Despite the vital role of guard cells in controlling plant water loss, ozone sensitivity and CO2 supply, the genes encoding some of the main regulators of stomatal movements remain unknown. It has been proposed that guard cell anion channels function as important regulators of stomatal closure and are essential in mediating stomatal responses to physiological and stress stimuli. However, the genes encoding membrane proteins that mediate guard cell anion efflux have not yet been identified. Here we report the mapping and characterization of an ozone-sensitive Arabidopsis thaliana mutant, slac1. We show that SLAC1 (SLOW ANION CHANNEL-ASSOCIATED 1) is preferentially expressed in guard cells and encodes a distant homologue of fungal and bacterial dicarboxylate/malic acid transport proteins. The plasma membrane protein SLAC1 is essential for stomatal closure in response to CO2, abscisic acid, ozone, light/dark transitions, humidity change, calcium ions, hydrogen peroxide and nitric oxide. Mutations in SLAC1 impair slow (S-type) anion channel currents that are activated by cytosolic Ca2+ and abscisic acid, but do not affect rapid (R-type) anion channel currents or Ca2+ channel function. A low homology of SLAC1 to bacterial and fungal organic acid transport proteins, and the permeability of S-type anion channels to malate suggest a vital role for SLAC1 in the function of S-type anion channels.

Rhesus glycoprotein gene expression in the mangrove killifishKryptolebias marmoratusexposed to elevated environmental ammonia levels and air

The mechanism(s) of ammonia excretion in the presence of elevated external ammonia are not well understood in fish. Recent studies in other organisms have revealed a new class of ammonia transporters, Rhesus glycoprotein genes (Rh genes), which may also play a role in ammonia excretion in fish. The first objective of this study was to clone and characterize Rh genes in a fish species with a relatively high tolerance to environmental ammonia, the mangrove killifish Kryptolebias marmoratus (formerly Rivulus marmoratus). We obtained full-length cDNAs of three Rh genes in K. marmoratus: RhBG (1736 bp), RhCG1 (1920 bp) and RhCG2 (2021 bp), which are highly homologous with other known Rh gene sequences. Hydropathy analysis revealed that all three Rh genes encode membrane proteins with 10-12 predicted transmembrane domains. RhBG, RhCG1 and RhCG2 are highly expressed in gill tissue, with RhBG also present in skin of K. marmoratus. Exposure to elevated environmental ammonia (2 mmol l(-1) NH(4)HCO(3)) for 5 days resulted in a modest (+37%) increase in whole-body ammonia levels, whereas gill RhCG2 and skin RhCG1 mRNA levels were upregulated by 5.8- and 7.7-fold, respectively. RhBG mRNA levels were also increased in various tissues, with 3- to 7-fold increases in the liver and skeletal muscle. In a separate group of killifish exposed to air for 24 h, RhCG1 and RhCG2 mRNA levels were elevated by 4- to 6-fold in the skin. Thus, the multifold induction of Rh mRNA levels in excretory tissues (gills and skin) and internal tissues in response to conditions that perturb normal ammonia excretion suggests that RhBG, RhCG1 and RhCG2 may be involved in facilitating ammonia transport in this species. Furthermore, the findings support earlier studies demonstrating that the skin is an important site of ammonia excretion in K. marmoratus.

Cloning and analysis of a novel conserved membrane zinc-metalloprotease family from Solanum surattense

Peptidases occur naturally in all organisms and their genes comprise 1–5% of the total number of genes. Genetic, biochemical, and molecular approaches used in recent years led to the identification and characterization of several plant organelle proteases, all of them being homologous to bacterial proteases best characterized in Escherichia coli. Here we report isolating and characterizing three novel genes, namely Sszn-mp1, Sszn-mp2, and SsZn-mp3 from Solanum surattense. To identify the subcellular location, structures, and functions of these three genes, integrated genomic approaches of data mining, expression profiling, and bioinformatic predictions were used. Sszn-mp is found to be constitutively expressed in tissues and regulated by various stimuli. Analysis of eight zinc-metalloproteases (Zn-MPs) deduced or assembled from Arabidopsis thaliana, tomato, potato, cotton, barley, sugarcane, and rice and four Zn-MPs from cyanobacteria (blue-green algae) in the GenBank database reveals that these proteins belong to a novel conserved membrane zinc-metalloprotease family. The plant Zn-MP members share more than 62% overall identity with SsZn-MP3, whereas four putative ATP-dependent zinc-proteases of cyanobacteria have low identity with SsZn-MP3 and their N-termini are about 110 amino acids shorter than those of plant Zn-MPs. The Zn-MP homologous sequences are found neither in other eukaryotic nor prokaryotic databases, suggesting that this family is specific to plants and cyanobacteria. The plant Zn-MP genes encoding membrane proteins are potentially targeted to chloroplast and plasma membranes, and the bacterial Zn-MPs are targeted to the cytoplasmic membrane, and their N-terminal targeting peptides are cleaved off for targeting the mature proteins to their subcellular compartments. The Zn-MP proteins contain a conserved zinc-binding site (HEAGHX19E/DX46∼48EX7E), a potential G-protein coupled receptors family 1 signature, and a triplet motif (N-R/K-F) in plant Zn-MPs, a D/E-R-Y motif in the four bacterial Zn-MPs, suggesting that the different mature forms of Zn-MPs may function as proteases and/or signal receptors.

Pichia pastoris is a valuable host for the expression of genes encoding membrane proteins from the hyperthermophilic Archeon Pyrococcus abyssi

We present here the experimental strategies, first results and identified bottlenecks of a structural genomics initiative on membrane proteins of the hyperthermophilic archaea Pyrococcus abyssi. Five ORFs coding for putative membrane proteins have been cloned and expressed in the methylotrophic Pichia pastoris expression system, using two different constructs, with or without the signal sequence alpha-mating factor of Saccharomyces cerevisiae. A c-myc epitope and 6 His codons were added at the 3'-end of the targeted genes to allow immunodetection of the recombinant proteins and to facilitate their further purification. We have selected at least one producer clone for each protein of interest and for almost every construction. All the membrane proteins were produced in Erlenmeyer flasks culture and in fed-batch cultivation for large-scale preparation. The proteins were detected in the membrane fractions of P. pastoris. Production efficiencies were relatively low in both production conditions but the quantities of biomass obtained during fed-batch cultivation have allowed us to collect sufficient amount of material for further purification. The proteins were extracted, solubilized and partially purified. Large-scale purification will be necessary for further structural work.

Identification and Expression Analysis of Twelve Members of the Nucleobase–Ascorbate Transporter (NAT) Gene Family in Arabidopsis thaliana

By screening genome databases, 12 genes encoding membrane proteins homologous to nucleobase-ascorbate transporters (NATs) were identified in Arabidopsis thaliana. A similar number of genes was found in the rice genome. The plant NAT proteins split into five clades (I-V) based on protein multisequence alignments. This classification nicely correlates with the patterns of organ- and tissue-specific expression during the whole life cycle of A. thaliana. Interestingly, expression of two members of clade III, AtNAT7 and AtNAT8, was found to be up-regulated in undifferentiated tissues such as callus or tumors produced by Agrobacterium tumefaciens. Clade V comprises AtNAT12 possessing a hydrophilic N-terminal extension. Transient expression of green fluorescent protein (GFP) fusions in different systems showed that AtNAT12 along with AtNAT7 and -8 are located in the plasma membrane. Mutations in any of the AtNAT genes do not induce phenotypic alterations. The absence of obvious mutant phenotypes in single but also in double and triple mutants suggests a high degree of functional redundancy between AtNAT genes, but might also point to redundant functions provided by genes or pathways unrelated to the AtNATs.

Transcript Profiles of Candida albicans Cortical Actin Patch Mutants Reflect Their Cellular Defects: Contribution of the Hog1p and Mkc1p Signaling Pathways

In Candida albicans, Myo5p and Sla2p are required for the polarized localization and function of cortical actin patches, for hyphal formation, and for endocytosis. Deletion of either the MYO5 or the SLA2 gene generated a common transcriptional response that involved changes in the transcript levels of cell wall protein- and membrane protein-encoding genes. However, these profiles were distinct from those observed for a mutant with specific deletions of the actin-organizing domains of Myo5p or for wild-type cells treated with cytochalasin A, both of which also generate defects in the organization of cortical actin patches. The profiles observed for the myo5Delta and sla2Delta mutants had similarities to those of wild-type cells subjected to an osmotic shock, and the defects in cortical patch function found with myo5Delta and sla2Delta mutants, but not cortical actin patch distribution per se, affected sensitivity to various stresses, including heat and osmotic shocks and cell wall damage. Secondary effects coupled with defective endocytosis, such as lack of polarized lipid rafts and associated protein Rvs167-GFP (where GFP is green fluorescent protein) and lack of polarized wall remodeling protein GFP-Gsc1, were also observed for the myo5Delta and sla2Delta mutants. The mitogen-activated protein kinases Hog1p and Mkc1p, which mediate signaling in response to osmotic stress and cell wall damage, do not play a major role in regulating the transcript level changes in the myo5Delta and sla2Delta mutants. Hog1p was not hyperphosphorylated in the myo5Delta and sla2Delta mutants, and the transcript levels of only a subset of genes affected in the myo5Delta mutant were dependent upon the presence of Hog1p and Mkc1p. However, it appears that Hog1p and Mkc1p play important roles in the myo5Delta mutant cells because double deletion of myosin I and either Hog1p or Mkc1p resulted in very-slow-growing cells.

Global Transcriptome Analysis ofTropheryma whippleiin Response to Temperature Stresses

Tropheryma whipplei, the agent responsible for Whipple disease, is a poorly known pathogen suspected to have an environmental origin. The availability of the sequence of the 0.92-Mb genome of this organism made a global gene expression analysis in response to thermal stresses feasible, which resulted in unique transcription profiles. A few genes were differentially transcribed after 15 min of exposure at 43 degrees C. The effects observed included up-regulation of the dnaK regulon, which is composed of six genes and is likely to be under control of two HspR-associated inverted repeats (HAIR motifs) found in the 5' region. Putative virulence factors, like the RibC and IspDF proteins, were also overexpressed. While it was not affected much by heat shock, the T. whipplei transcriptome was strongly modified following cold shock at 4 degrees C. For the 149 genes that were differentially transcribed, eight regulons were identified, and one of them was composed of five genes exhibiting similarity with genes encoding ABC transporters. Up-regulation of these genes suggested that there was an increase in nutrient uptake when the bacterium was exposed to cold stress. As observed for other bacterial species, the major classes of differentially transcribed genes encode membrane proteins and enzymes involved in fatty acid biosynthesis, indicating that membrane modifications are critical. Paradoxically, the heat shock proteins GroEL2 and ClpP1 were up-regulated. Altogether, the data show that despite the lack of classical regulation pathways, T. whipplei exhibits an adaptive response to thermal stresses which is consistent with its specific environmental origin and could allow survival under cold conditions.

Global Transcriptome Analysis of the Cold Shock Response of Shewanellaoneidensis MR-1 and Mutational Analysis of Its ClassicalCold ShockProteins

This study presents a global transcriptional analysis of the cold shock response of Shewanella oneidensis MR-1 after a temperature downshift from 30 degrees C to 8 or 15 degrees C based on time series microarray experiments. More than 700 genes were found to be significantly affected (P < or = 0.05) upon cold shock challenge, especially at 8 degrees C. The temporal gene expression patterns of the classical cold shock genes varied, and only some of them, most notably so1648 and so2787, were differentially regulated in response to a temperature downshift. The global response of S. oneidensis to cold shock was also characterized by the up-regulation of genes encoding membrane proteins, DNA metabolism and translation apparatus components, metabolic proteins, regulatory proteins, and hypothetical proteins. Most of the metabolic proteins affected are involved in catalytic processes that generate NADH or NADPH. Mutational analyses confirmed that the small cold shock proteins, So1648 and So2787, are involved in the cold shock response of S. oneidensis. The analyses also indicated that So1648 may function only at very low temperatures.