Psychrophilic <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> requires <Emphasis Type="Italic">trans</Emphasis>-monounsaturated fatty acid for growth at higher temperature

A psychrophilic bacterium, Pseudomonas syringae (Lz4W) from Antarctica, was used as a model system to establish a correlation, if any, between thermal adaptation, trans-fatty acid content and membrane fluidity. In addition, attempts were made to clone and sequence the cti gene of P. syringae (Lz4W) so as to establish its characteristics with respect to the cti of other Pseudomonas spp. and also to in vitro mutagenize the cti gene so as to generate a cti null mutant. The bacterium showed increased proportion of saturated and trans-monounsaturated fatty acids when grown at 28°C compared to cells grown at 5°C, and the membrane fluidity decreased with growth temperature. In the mutant, the trans-fatty acid was not synthesized, and the membrane fluidity also decreased with growth temperature, but the decrease was not to the extent that was observed in the wild-type cells. Thus, it would appear that synthesis of trans-fatty acid and modulation of membrane fluidity to levels comparable to the wild-type cells is essential for growth at higher temperatures since the mutant exhibits growth arrest at 28°C. In fact, the cti null mutant-complemented strain of P. syringae (Lz4W-C30b) that was capable of synthesizing the trans-fatty acid was indeed capable of growth at 28°C, thus confirming the above contention. The cti gene of P. syringae (Lz4W) that was cloned and sequenced exhibited high sequence identity with the cti of other Pseudomonas spp. and exhibited all the conserved features.     

<Emphasis Type="Italic">Physcomitrella patens</Emphasis> is highly tolerant against drought,salt and osmotic stress

In order to determine the degree of tolerance of the moss Physcomitrella patens to different abiotic stress conditions, we examined its tolerance against salt, osmotic and dehydration stress. Compared to other plants like Arabidopsis thaliana, P. patens exhibits a high degree of abiotic stress tolerance, making it a valuable source for the identification of genes effecting the stress adaptation. Plants that had been treated with NaCl tolerated concentrations up to 350 mM. Treatments with sorbitol revealed that plants are able to survive concentrations up to 500 mM. Furthermore, plants that had lost 92% water on a fresh-weight basis were able to recover successfully. For molecular analyses, a P. patens expressed sequence tag (EST) database was searched for cDNA sequences showing homology to stress-associated genes of seed plants and bacteria. 45 novel P. patens genes were identified and subjected to cDNA macroarray analyses to define their expression pattern in response to water deficit. Among the selected cDNAs, we were able to identify a set of genes that is specifically up-regulated upon dehydration. These genes encode proteins exerting their function in maintaining the integrity of the plant cell as well as proteins that are known to be members of signaling networks. The identified genes will serve as molecular markers and potential targets for future functional analyses.     

Identification and characterisation of <Emphasis Type="Italic">ssrA</Emphasis> in members of the <Emphasis Type="Italic">Helicobacter</Emphasis> genus

Bacterial ssrA encodes tmRNA that functions both as a tRNA and an mRNA to rescue the stalled ribosome on defective mRNAs. In this study, ssrA was identified in four gastric species of Helicobacters and four enterohepatic species of Helicobacters. The tag peptide of 14 amino acids encoded by ssrA showed a pattern of Val(1)Ala(13) in gastric species, a pattern of Ala(1)Val(13 )in enterohepatic species, in contrast to the pattern of Ala(1)Ala(13) in W. succinogenes and C. jejuni, which are closely related to helicobacters. Phylogenetic analysis and the patterns of the tag peptide suggest that the Helicobacter genus could be separated into two genera. High conservation of ssrA in H. pylori was observed. The annotated ORF HP0784 in H. pylori, which largely overlaps ssrA, is unlikely to be functional. H. pylori ssrA interestingly expressed a large and a small tmRNA molecule.     

Structure and allele-specific expression variation of novel α/β hydrolase fold proteins in gentian plants

Previously, we identified two closely related proteins termed W14 and W15 that were enriched in the overwinter buds of the gentian plant Gentiana triflora. Expression of the latter protein W15 has been implicated in its association with cold hardiness, because of its absence in a cold-sensitive mutant. Here, we characterized these two proteins and the genes encoding them. Amino acid sequences of the W14 and W15 proteins showed difference at only three amino acid positions, and both of them showed homologies to α/β hydrolase fold superfamily. Consistently, GST-fused W14 and W15 proteins expressed in bacteria showed hydrolase activity toward 1-naphtyl acetate. Structural analysis of these two genes in seven different gentian strains/cultivars including an anther culture-derived homozygous diploid revealed that W14 and W15 genes are allelic. Three genotypes were found; two strains carried both alleles (W14/W15), one carried the W15 genes in both alleles (W15/W15), and others were homozygous of W14 (W14/W14). Interestingly, expression of the two proteins exhibited allele-specificity. In one W14/W15 strain, expression of the W15 allele was almost repressed. In addition, organ specific expression of the alleles was observed in different cultivars. These observations were discussed in relation to winter hardiness of the gentian plants. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB281493 and AB281494.     

Novel molecular features of the fibrolytic intestinal bacterium Fibrobacter intestinalis not shared with Fibrobacter succinogenes as determined by suppressive subtractive hybridization

Suppressive subtractive hybridization was conducted to identify unique genes coding for plant cell wall hydrolytic enzymes and other properties of the gastrointestinal bacterium Fibrobacter intestinalis DR7 not shared by Fibrobacter succinogenes S85. Subtractive clones from F. intestinalis were sequenced and assembled to form 712 nonredundant contigs with an average length of 525 bp. Of these, 55 sequences were unique to F. intestinalis. The remaining contigs contained 764 genes with BLASTX similarities to other proteins; of these, 80% had the highest similarities to proteins in F. succinogenes, including 30 that coded for carbohydrate active enzymes. The expression of 17 of these genes was verified by Northern dot blot analysis. Of genes not exhibiting BLASTX similarity to F. succinogenes, 30 encoded putative transposases, 6 encoded restriction modification genes, and 45% had highest similarities to proteins in other species of gastrointestinal bacteria, a finding suggestive of either horizontal gene transfer to F. intestinalis or gene loss from F. succinogenes. Analysis of contigs containing segments of two or more adjacent genes revealed that only 35% exhibited BLASTX similarity and were in the same orientation as those of F. succinogenes, indicating extensive chromosomal rearrangement. The expression of eight transposases, and three restriction-modification genes was confirmed by Northern dot blot analysis. These data clearly document the maintenance of carbohydrate active enzymes in F. intestinalis necessitated by the preponderance of polysaccharide substrates available in the ruminal environment. It also documents substantive changes in the genome from that of F. succinogenes, which may be related to the introduction of the array of transposase and restriction-modification genes.     

Identification of an anaerobic bacterium which reduces perchlorate and chlorate asWolinella succinogenes

A bacterium coded as strain HAP-1 was isolated from a municipal anaerobic digestor for its ability to reduce >7000 ppm perchlorate in wastewaters. The organism is capable of the dissimilatory reduction of perchlorate on chlorate to chloride for energy and growth. It is a Gram-negative, non-sporeforming, obligately anaerobic, motile thin rod. Antibiotic resistance, utilization of carbon substrates and utilization of electron acceptors by bacterium HAP-1 were similar toWolinella succinogenes. The organism's 16S rRNA sequence was 0.75% different from that of the type strain ofW. succinogenes. The fatty acid compositions of the two organisms are very similar. The morphological, physiological and 16S rRNA sequence data indicated that bacterium HAP-1 is a strain ofW. succinogenes that can utilize perchlorate or chlorate as a terminal electron acceptor.     

Comparative proteome analysis of robust <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> insights into industrial continuous and batch fermentation

A robust Saccharomyces cerevisiae strain has been widely applied in continuous and batch/fed-batch industrial fermentation. However, little is known about the molecular basis of fermentative behavior of this strain in the two realistic fermentation processes. In this paper, we presented comparative proteomic profiling of the industrial yeast in the industrial fermentation processes. The expression levels of most identified protein were closely interrelated with the different stages of fermentation processes. Our results indicate that, among the 47 identified protein spots, 17 of them belonging to 12 enzymes were involved in pentose phosphate, glycolysis, and gluconeogenesis pathways and glycerol biosynthetic process, indicating that a number of pathways will need to be inactivated to improve ethanol production. The differential expressions of eight oxidative response and heat-shock proteins were also identified, suggesting that it is necessary to keep the correct cellular redox or osmotic state in the two industrial fermentation processes. Moreover, there are significant differences in changes of protein levels between the two industrial fermentation processes, especially these proteins associated with the glycolysis and gluconeogenesis pathways. These findings provide a molecular understanding of physiological adaptation of industrial strain for optimizing the performance of industrial bioethanol fermentation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Heterologous overexpression of quorum-sensing regulators to study cell-density-dependent phenotypes in a symbiotic plant bacterium <Emphasis Type="Italic">Mesorhizobium huakuii</Emphasis>

Quorum-sensing is widespread among many prokaryotic lineages. In order to investigate quorum regulation in the plant bacterium Mesorhizobium huakuii which produces an N-acyl homoserine lactone (AHL) quorum signal, the Agrobacterium quorum-sensing regulator TraR was heterologously expressed in this bacterium. The resulting strains showed reduced AHL production in the supernatant compared to wild-type, but similar intracellular levels of AHLs were detected, suggesting that M. huakuii AHLs can be bound to intracellular TraR proteins and thus become unavailable for its own quorum systems. M. huakuii overexpressing TraR formed thinner biofilms than the wild-type, suggesting a role played by quorum-sensing in biofilm formation.Hui Wang and Zengtao Zhong contributed equally to this work.     

<Emphasis Type="Italic">Cupriavidus metallidurans</Emphasis>: evolution of a metal-resistant bacterium

Cupriavidus metallidurans CH34 has gained increasing interest as a model organism for heavy metal detoxification and for biotechnological purposes. Resistance of this bacterium to transition metal cations is predominantly based on metal resistance determinants that contain genes for RND (resistance, nodulation, and cell division protein family) proteins. These are part of transenvelope protein complexes, which seem to detoxify the periplasm by export of toxic metal cations from the periplasm to the outside. Strain CH34 contains 12 predicted RND proteins belonging to a protein family of heavy metal exporters. Together with many efflux systems that detoxify the cytoplasm, regulators and possible metal-binding proteins, RND proteins mediate an efficient defense against transition metal cations. To shed some light into the origin of genes encoding these proteins, the genomes of C. metallidurans CH34 and six related proteobacteria were investigated for occurrence of orthologous and paralogous proteins involved in metal resistance. Strain CH34 was not much different from the other six bacteria when the total content of transport proteins was compared but CH34 had significantly more putative transition metal transport systems than the other bacteria. The genes for these systems are located on its chromosome 2 but especially on plasmids pMOL28 and pMOL30. Cobalt–nickel and chromate resistance determinants located on plasmid pMOL28 evolved by gene duplication and horizontal gene transfer events, leading to a better adaptation of strain CH34 to serpentine-like soils. The czc cobalt–zinc–cadmium resistance determinant, located on plasmid pMOL30 in addition copper, lead and mercury resistance determinants, arose by duplication of a czcICAB core determinant on chromosome 2, plus addition of the czcN gene upstream and the genes czcD, czcRS, czcE downstream of czcICBA. C. metallidurans apparently evolved metal resistance by horizontal acquisition and by duplication of genes for transition metal efflux, mostly on the two plasmids, and decreased the number of uptake systems for those metals. This paper is dedicated to Dr. Max Mergeay for a long time of cooperation, constructive competition and friendship.     

Mass spectrometry proteomic analysis of stress adaptation reveals both common and distinct response pathways in<Emphasis Type="Italic"> Propionibacterium freudenreichii</Emphasis>

Microorganisms used in food technology and probiotics are exposed to technological and digestive stresses, respectively. Traditionally used as Swiss-type cheese starters, propionibacteria also constitute promising human probiotics. Stress tolerance and cross-protection in Propionibacterium freudenreichii were thus examined after exposure to heat, acid, or bile salts stresses. Adapted cells demonstrated acquired homologous tolerance. Cross-protection between bile salts and heat adaptation was demonstrated. By contrast, bile salts pretreatment sensitized cells to acid challenge and vice versa. Surprisingly, heat and acid responses did not present significant cross-protection in P. freudenreichii. During adaptations, important changes in cellular protein synthesis were observed using two-dimensional electrophoresis. While global protein synthesis decreased, several proteins were overexpressed during stress adaptations. Thirty-four proteins were induced by acid pretreatment, 34 by bile salts pretreatment, and 26 by heat pretreatment. Six proteins are common to all stresses and represent general stress-response components. Among these polypeptides, general stress chaperones, and proteins involved in energetic metabolism, oxidative stress response, or SOS response were identified. These results bring new insight into the tolerance of P. freudenreichii to heat, acid, and bile salts, and should be taken into consideration in the development of probiotic preparations.     

Detection of a <Emphasis Type="Italic">SERK</Emphasis>-like gene in coconut and analysis of its expression during the formation of embryogenic callus and somatic embryos

Somatic embryogenesis involves different molecular events including differential gene expression and various signal transduction pathways. One of the genes identified in early somatic embryogenesis is S OMATIC E MBRYOGENESIS R ECEPTOR-like K INASE (SERK). Cocos nucifera (L.) is one of the most recalcitrant species for in vitro regeneration, achieved so far only through somatic embryogenesis, although just a few embryos could be obtained from a single explant. In order to increase efficiency of this process we need to understand it better. Therefore, the purpose of the present work was to determine if an ortholog of the SERK gene is present in the coconut genome, isolate it and analyze its expression during somatic embryogenesis. The results showed the occurrence of a SERK ortholog referred to as CnSERK. Predicted sequence analysis showed that CnSERK encodes a SERK protein with the domains reported in the SERK proteins in other species. These domains consist of a signal peptide, a leucine zipper domain, five LRR, the Serine-Proline-Proline domain, which is a distinctive domain of the SERK proteins, a single transmembrane domain, the kinase domain with 11 subdomains and the C terminal region. Analysis of its expression showed that it could be detected in embryogenic tissues before embryo development could be observed. In contrast it was not detected or at lower levels in non-embryogenic tissues, thus suggesting that CnSERK expression is associated with induction of somatic embryogenesis and that it could be a potential marker of cells competent to form somatic embryos in coconut tissues cultured in vitro.