Extracytoplasmic Space Research Articles

Transmembrane formins as active cargoes of membrane trafficking.

Formins are a large, evolutionarily old family of cytoskeletal regulators whose roles include actin capping and nucleation, as well as modulation of microtubule dynamics. The plant class I formin clade is characterized by a unique domain organization, as most of its members are transmembrane proteins with possible cell wall-binding motifs exposed to the extracytoplasmic space-a structure that appears to be a synapomorphy of the plant kingdom. While such transmembrane formins are traditionally considered mainly as plasmalemma-localized proteins contributing to the organization of the cell cortex, we review, from a cell biology perspective, the growing evidence that they can also, at least temporarily, reside (and in some cases also function) in endomembranes including secretory and endocytotic pathway compartments, the endoplasmic reticulum, the nuclear envelope, and the tonoplast. Based on this evidence, we propose that class I formins may thus serve as 'active cargoes' of membrane trafficking-membrane-embedded proteins that modulate the fate of endo- or exocytotic compartments while being transported by them.

HtrAs are essential for the survival of the haloarchaeon Natrinema gari J7-2 in response to heat, high salinity, and toxic substances.

Bacterial and eukaryotic HtrAs can act as an extracytoplasmic protein quality control (PQC) system to help cells survive in stress conditions, but the functions of archaeal HtrAs remain unknown. Particularly, haloarchaea route most secretory proteins to the Tat pathway, enabling them to fold properly in well-controlled cytoplasm with cytosolic PQC systems before secretion. It is unclear whether HtrAs are required for haloarchaeal survival and stress response. The haloarchaeon Natrinema gari J7-2 encodes three Tat signal peptide-bearing HtrAs (NgHtrA, NgHtrB, and NgHtrC), and the signal peptides of NgHtrA and NgHtrC contain a lipobox. Here, the in vitro analysis reveals that the three HtrAs show different profiles of temperature-, salinity-, and metal ion-dependent proteolytic activities and could exhibit chaperone-like activities to prevent the aggregation of reduced lysozyme when their proteolytic activities are inhibited at low temperatures or the active site is disrupted. The gene deletion and complementation assays reveal that NgHtrA and NgHtrC are essential for the survival of strain J7-2 at elevated temperature and/or high salinity and contribute to the resistance of this haloarchaeon to zinc and inhibitory substances generated from tryptone. Mutational analysis shows that the lipobox mediates membrane anchoring of NgHtrA or NgHtrC, and both the membrane-anchored and free extracellular forms of the two enzymes are involved in the stress resistance of strain J7-2, depending on the stress conditions. Deletion of the gene encoding NgHtrB in strain J7-2 causes no obvious growth defect, but NgHtrB can functionally substitute for NgHtrA or NgHtrC under some conditions.IMPORTANCEHtrA-mediated protein quality control plays an important role in the removal of aberrant proteins in the extracytoplasmic space of living cells, and the action mechanisms of HtrAs have been extensively studied in bacteria and eukaryotes; however, information about the function of archaeal HtrAs is scarce. Our results demonstrate that three HtrAs of the haloarchaeon Natrinema gari J7-2 possess both proteolytic and chaperone-like activities, confirming that the bifunctional nature of HtrAs is conserved across all three domains of life. Moreover, we found that NgHtrA and NgHtrC are essential for the survival of strain J7-2 under stress conditions, while NgHtrB can serve as a substitute for the other two HtrAs under certain circumstances. This study provides the first biochemical and genetic evidence of the importance of HtrAs for the survival of haloarchaea in response to stresses.

SecA - a multidomain and multitask bacterial export protein.

Most bacterial secretory proteins destined to the extracytoplasmic space are secreted posttranslationally by the Sec translocase. SecA, a key component of the Sec system, is the ATPase motor protein, directly responsible for transferring the preprotein across the cytoplasmic membrane. SecA is a large protein, composed of several domains, capable of binding client preproteins and a variety of partners, including the SecYEG inner membrane channel complex, membrane phospholipids and ribosomes. SecA-mediated translocation can be divided into two major steps: (1) targeting of the preproteins to the membrane translocation apparatus and (2) transport across the membrane through the SecYEG channel. In this review we present current knowledge regarding SecA structure and function of this protein in both translocation steps. The most recent model of the SecA-dependent preprotein mechanical translocation across the bacterial cytoplasmic membrane is described. A possibility of targeting SecA with inhibitory compounds as a strategy to combat pathogenic bacteria will be discussed as well.

Extracytoplasmic diaphorase activity of Streptomyces coelicolor A3(2)

Metabolism and utilization of plant-derived aromatic substances are fundamental to the saprophytic growth of Streptomyces. Here, we studied an enzyme activity reducing 2,6-dichlorophenolindophenol and nitroblue tetrazolium in the culture supernatant of Streptomyces coelicolor A3(2). N-terminal amino acid sequencing of a nitroblue tetrazolium-reducing enzyme revealed that the enzyme corresponds to the SCO2180 product. The protein exhibited a marked similarity with dihydrolipoamide dehydrogenase, the E3 subunit of 2-oxo-acid dehydrogenase complex. A recombinant SCO2180 protein formed a homodimer and exhibited a diaphorase activity catalyzing NADH-dependent reduction of various quinonic substrates. Similar nitroblue tetrazolium-reducing activities were observed for other Streptomyces strains isolated from soil, implying that the diaphorase-catalyzed reduction of quinonic substances widely occurs in the extracytoplasmic space of Streptomyces.

Antibiotic Resistance Mediated by the MacB ABC Transporter Family: A Structural and Functional Perspective.

The MacB ABC transporter forms a tripartite efflux pump with the MacA adaptor protein and TolC outer membrane exit duct to expel antibiotics and export virulence factors from Gram-negative bacteria. Here, we review recent structural and functional data on MacB and its homologs. MacB has a fold that is distinct from other structurally characterized ABC transporters and uses a unique molecular mechanism termed mechanotransmission. Unlike other bacterial ABC transporters, MacB does not transport substrates across the inner membrane in which it is based, but instead couples cytoplasmic ATP hydrolysis with transmembrane conformational changes that are used to perform work in the extra-cytoplasmic space. In the MacAB-TolC tripartite pump, mechanotransmission drives efflux of antibiotics and export of a protein toxin from the periplasmic space via the TolC exit duct. Homologous tripartite systems from pathogenic bacteria similarly export protein-like signaling molecules, virulence factors and siderophores. In addition, many MacB-like ABC transporters do not form tripartite pumps, but instead operate in diverse cellular processes including antibiotic sensing, cell division and lipoprotein trafficking.

Disulfide bond formation in prokaryotes.

Interest in protein disulfide bond formation has recently increased because of the prominent role of disulfide bonds in bacterial virulence and survival. The first discovered pathway that introduces disulfide bonds into cell envelope proteins consists of Escherichia coli enzymes DsbA and DsbB. Since its discovery, variations on the DsbAB pathway have been found in bacteria and archaea, probably reflecting specific requirements for survival in their ecological niches. One variation found amongst Actinobacteria and Cyanobacteria is the replacement of DsbB by a homologue of human vitamin K epoxide reductase. Many Gram-positive bacteria express enzymes involved in disulfide bond formation that are similar, but non-homologous, to DsbAB. While bacterial pathways promote disulfide bond formation in the bacterial cell envelope, some archaeal extremophiles express proteins with disulfide bonds both in the cytoplasm and in the extra-cytoplasmic space, possibly to stabilize proteins in the face of extreme conditions, such as growth at high temperatures. Here, we summarize the diversity of disulfide-bond-catalysing systems across prokaryotic lineages, discuss examples for understanding the biological basis of such systems, and present perspectives on how such systems are enabling advances in biomedical engineering and drug development.

Structure of the ectodomain of the electron transporter Rv2874 from Mycobacterium tuberculosis reveals a thioredoxin-like domain combined with a carbohydrate-binding module.

The members of the CcdA family are integral membrane proteins that use a disulfide cascade to transport electrons from the thioredoxin-thioredoxin reductase system in the interior of the cell into the extracytoplasmic space. The core transmembrane portion of this family is often elaborated with additional hydrophilic domains that act as adapters to deliver reducing potential to targets outside the cellular membrane. To investigate the function of family members in Mycobacterium tuberculosis, the structure of the C-terminal ectodomain from Rv2874, one of three CcdA-family members present in the genome, was determined. The crystal structure, which was refined at 1.9 Å resolution with R = 0.195 and Rfree = 0.219, reveals the predicted thioredoxin-like domain with its conserved Cys-X-X-Cys active-site motif. Unexpectedly, this domain is combined with a second domain with a carbohydrate-binding module (CBM) fold, this being the first reported example of a CBM in association with a thioredoxin-like domain fold. A cavity in the CBM adjacent to the thioredoxin active site suggests a likely carbohydrate-binding site, representing a broadening of the substrate range for CcdA-family members and an expansion of the thioredoxin-domain functionality to carbohydrate modification.

N-terminal transmembrane domain of lipase LipA from Pseudomonas protegens Pf-5: A must for its efficient folding into an active conformation.

LipA from Pseudomonas protegens Pf-5 has been proven not to be secreted into the extracytoplasmic space, proposing that it is a membrane protein in virtue of its N-terminal transmembrane domain predicted by the TMHMM 2.0. However, LipA was confirmed to be an intracellular protein through determining the effects of lipA deletion or overexpression on the lipase activities in the whole-cell, lysis supernatant and lysis pellet, even through its transmembrane domain being able to make heterologous LacZ locate on the cytoplasmic membrane via construction of β-galactosidase reporter strains. Subsequently, lipase activity assays showed that the transmembrane domain played an indispensable role for the catalytic function of LipA through construction of the markerless deletion mutant of transmembrane domain sequence of lipA and the expression and purification of LipA and LipAΔTMD. To further investigate why the transmembrane domain lost its membrane localization function and significantly affected the catalytic function of LipA, the 3D structures of LipA and LipAΔTMD were constructed. The results indicated that the transmembrane domain, located in the interior of LipA, helped the α-helical lid to form an open conformation by the mediation of α5 helix. It seems to act as a kind of intramolecular chaperone like the β-roll motif of subfamily I.3 lipases, which is novel and is the first to notify the intramolecular chaperone of a subfamily I.1 lipase.

The MprB Extracytoplasmic Domain Negatively Regulates Activation of the Mycobacterium tuberculosis MprAB Two-Component System

Mycobacterium tuberculosis is an acid-fast pathogen of humans and the etiological agent of tuberculosis (TB). It is estimated that one-third of the world's population is latently (persistently) infected with M. tuberculosis. M. tuberculosis persistence is regulated, in part, by the MprAB two-component signal transduction system, which is activated by and mediates resistance to cell envelope stress. Here we identify MprAB as part of an evolutionarily conserved cell envelope stress response network and demonstrate that MprAB-mediated signal transduction is negatively regulated by the MprB extracytoplasmic domain (ECD). In particular, we report that deregulated production of the MprB sensor kinase, or of derivatives of this protein, negatively impacts M. tuberculosis growth. The observed growth attenuation is dependent on MprAB-mediated signal transduction and is exacerbated in strains of M. tuberculosis producing an MprB variant lacking its ECD. Interestingly, full-length MprB, and the ECD of MprB specifically, immunoprecipitates the Hsp70 chaperone DnaK in vivo, while overexpression of dnaK inhibits MprAB-mediated signal transduction in M. tuberculosis grown in the absence or presence of cell envelope stress. We propose that under nonstress conditions, or under conditions in which proteins present in the extracytoplasmic space are properly folded, signaling through the MprAB system is inhibited by the MprB ECD. Following exposure to cell envelope stress, proteins present in the extracytoplasmic space become unfolded or misfolded, leading to removal of the ECD-mediated negative regulation of MprB and subsequent activation of MprAB.

Co-expression analysis, proteomic and metabolomic study on the impact of a Deg/HtrA protease triple mutant in Synechocystis sp. PCC 6803 exposed to temperature and high light stress

Members of the DegP/HtrA protease family are widespread in nature and play an important role in proteolysis of misfolded and damaged proteins. The cyanobacterium Synechocystis sp. PCC 6803 contains three Deg proteases, HhoA (Sll1679), HhoB (Sll1427) and HtrA (Slr1204). Using the proteomic or metabolomic approach we investigated a triple deletion mutant (Δdeg) exposed to light or temperature stress. To cope with the stress conditions the triple mutant reduces its energy metabolism and stress-related proteins are induced to protect the cells. Additionally the co-expression of the genes encoding the three proteases with other genes in Synechocystis sp. PCC 6803 was analyzed. While HhoA seems to be involved in house-keeping processes related to protein (re)folding, protein clearance and signaling, the hhoB expression cluster is dominated by genes encoding periplasmic proteins linked to metabolism or signal transduction pathways. The htrA expression pattern is similar to that of genes encoding proteins of the electron transport chain, iron- and nitrogen metabolism. Our integrative approach indicates significant rearrangements in cells depleted of the Deg/HtrA proteases when exposed to stress, both, in the cytoplasmic and extracytoplasmic space.

A Novel Membrane Protein, VanJ, Conferring Resistance to Teicoplanin

Bacterial resistance to the glycopeptide antibiotic teicoplanin shows some important differences from the closely related compound vancomycin. They are currently poorly understood but may reflect significant differences in the mode of action of each antibiotic. Streptomyces coelicolor possesses a vanRSJKHAX gene cluster that when expressed confers resistance to both vancomycin and teicoplanin. The resistance to vancomycin is mediated by the enzymes encoded by vanKHAX, but not by vanJ. vanHAX effect a reprogramming of peptidoglycan biosynthesis, which is considered to be generic, conferring resistance to all glycopeptide antibiotics. Here, we show that vanKHAX are not in fact required for teicoplanin resistance in S. coelicolor, which instead is mediated solely by vanJ. vanJ is shown to encode a membrane protein oriented with its C-terminal active site exposed to the extracytoplasmic space. VanJ also confers resistance to the teicoplanin-like antibiotics ristocetin and A47934 and to a broad range of semisynthetic teicoplanin derivatives, but not generally to antibiotics or semisynthetic derivatives with vancomycin-like structures. vanJ homologues are found ubiquitously in streptomycetes and include staP from the Streptomyces toyocaensis A47934 biosynthetic gene cluster. While overexpression of staP also conferred resistance to teicoplanin, similar expression of other vanJ homologues (SCO2255, SCO7017, and SAV5946) did not. The vanJ and staP orthologues, therefore, appear to represent a subset of a larger protein family whose members have acquired specialist roles in antibiotic resistance. Future characterization of the divergent enzymatic activity within this new family will contribute to defining the molecular mechanisms important for teicoplanin activity and resistance.

Mycobacterium tuberculosis MycP1 Protease Plays a Dual Role in Regulation of ESX-1 Secretion and Virulence

Mycobacterium tuberculosis uses the ESX-1 secretion system to deliver virulence proteins during infection of host cells. Here we report a mechanism of posttranscriptional control of ESX-1 mediated by MycP1, a M. tuberculosis serine protease. We show that MycP1 is required for ESX-1 secretion but that, unexpectedly, genetic inactivation of MycP1 protease activity increases secretion of ESX-1 substrates. We demonstrate that EspB, an ESX-1 substrate required for secretion, is a target of MycP1 in vitro and in vivo. During macrophage infection, an inactive MycP1 protease mutant causes hyperactivation of ESX-1-stimulated innate signaling pathways. MycP1 is required for growth in mice during acute infection, while loss of its protease activity leads to attenuated virulence during chronic infection. As the key ESX-1 substrates ESAT-6 and CFP-10 are highly immunogenic, fine-tuning of their secretion by MycP1 may balance virulence and immune detection and be essential for successful maintenance of long-term M. tuberculosis infection.

Identification of an Atypical Membrane Protein Involved in the Formation of Protein Disulfide Bonds in Oxygenic Photosynthetic Organisms

The evolution of oxygenic photosynthesis in cyanobacteria nearly three billion years ago provided abundant reducing power and facilitated the elaboration of numerous oxygen-dependent reactions in our biosphere. Cyanobacteria contain an internal thylakoid membrane system, the site of photosynthesis, and a typical Gram-negative envelope membrane system. Like other organisms, the extracytoplasmic space in cyanobacteria houses numerous cysteine-containing proteins. However, the existence of a biochemical system for disulfide bond formation in cyanobacteria remains to be determined. Extracytoplasmic disulfide bond formation in non-photosynthetic organisms is catalyzed by coordinated interaction between two proteins, a disulfide carrier and a disulfide generator. Here we describe a novel gene, SyndsbAB, required for disulfide bond formation in the extracytoplasmic space of cyanobacteria. The SynDsbAB orthologs are present in most cyanobacteria and chloroplasts of higher plants with fully sequenced genomes. The SynDsbAB protein contains two distinct catalytic domains that display significant similarity to proteins involved in disulfide bond formation in Escherichia coli and eukaryotes. Importantly, SyndsbAB complements E. coli strains defective in disulfide bond formation. In addition, the activity of E. coli alkaline phosphatase localized to the periplasm of Synechocystis 6803 is dependent on the function of SynDsbAB. Deletion of SyndsbAB in Synechocystis 6803 causes significant growth impairment under photoautotrophic conditions and results in hyper-sensitivity to dithiothreitol, a reductant, whereas diamide, an oxidant had no effect on the growth of the mutant strains. We conclude that SynDsbAB is a critical protein for disulfide bond formation in oxygenic photosynthetic organisms and required for their optimal photoautotrophic growth.

Evidence that the COOH terminus of human presenilin 1 is located in extracytoplasmic space

The polytopic membrane protein presenilin 1 (PS1) is a component of the gamma-secretase complex that is responsible for the intramembranous cleavage of several type I transmembrane proteins, including the beta-amyloid precursor protein (APP). Mutations of PS1, apparently leading to aberrant processing of APP, have been genetically linked to early-onset familial Alzheimer's disease. PS1 contains 10 hydrophobic regions (HRs) sufficiently long to be alpha-helical membrane spanning segments. Most topology models for PS1 place its COOH terminal approximately 40 amino acids, which include HR 10, in the cytosolic space. However, several recent observations suggest that HR 10 may be integrated into the membrane and involved in the interaction between PS1 and APP. We have applied three independent methodologies to investigate the location of HR 10 and the extreme COOH terminus of PS1. The results from these methods indicate that HR 10 spans the membrane and that the COOH terminal amino acids of PS1 lie in the extracytoplasmic space.

Interactions among Rax1p, Rax2p, Bud8p, and Bud9p in marking cortical sites for bipolar bud-site selection in yeast.

In the budding yeast Saccharomyces cerevisiae, selection of the bud site determines the axis of polarized cell growth and eventual oriented cell division. Bud sites are selected in specific patterns depending on cell type. These patterns appear to depend on distinct types of marker proteins in the cell cortex; in particular, the bipolar budding of diploid cells depends on persistent landmarks at the birth-scar-distal and -proximal poles that involve the proteins Bud8p and Bud9p, respectively. Rax1p and Rax2p also appear to function specifically in bipolar budding, and we report here a further characterization of these proteins and of their interactions with Bud8p and Bud9p. Rax1p and Rax2p both appear to be integral membrane proteins. Although commonly used programs predict different topologies for Rax2p, glycosylation studies indicate that it has a type I orientation, with its long N-terminal domain in the extracytoplasmic space. Analysis of rax1 and rax2 mutant budding patterns indicates that both proteins are involved in selecting bud sites at both the distal and proximal poles of daughter cells as well as near previously used division sites on mother cells. Consistent with this, GFP-tagged Rax1p and Rax2p were both observed at the distal pole as well as at the division site on both mother and daughter cells; localization to the division sites was persistent through multiple cell cycles. Localization of Rax1p and Rax2p was interdependent, and biochemical studies showed that these proteins could be copurified from yeast. Bud8p and Bud9p could also be copurified with Rax1p, and localization studies provided further evidence of interactions. Localization of Rax1p and Rax2p to the bud tip and distal pole depended on Bud8p, and normal localization of Bud8p was partially dependent on Rax1p and Rax2p. Although localization of Rax1p and Rax2p to the division site did not appear to depend on Bud9p, normal localization of Bud9p appeared largely or entirely dependent on Rax1p and Rax2p. Taken together, the results indicate that Rax1p and Rax2p interact closely with each other and with Bud8p and Bud9p in the establishment and/or maintenance of the cortical landmarks for bipolar budding.

The serine protease HtrA1 is upregulated in the human placenta during pregnancy.

The placenta has a dynamic and continuous capacity for self-renewal. The molecular mechanisms responsible for controlling trophoblast proliferation are still unclear. It is generally accepted that the simultaneous activity of proteins involved in cell proliferation, apoptosis, and extracellular matrix degradation plays an important role in correct placental development. We investigated in depth the expression of the serine protease HtrA1 during pregnancy in human placenta by in situ hybridization and immunohistochemistry, we demonstrated that HtrA1 displayed a low level of expression in the first trimester of gestation and a strong increase of HtrA1 expression in the third trimester. Finally, by electron microscopy, we demonstrated that HtrA1 was localized either in the cytoplasm of placental cells, especially close to microvilli that characterized the plasma membrane of syncytiotrophoblast cells, or in the extracytoplasmic space of the stroma of placental villi, particularly in the spaces between collagen fibers and on collagen fibers themselves. The expression pattern of HtrA1 in human placentas strongly suggests a role for this protein in placental development and function. Moreover, on the basis of its subcellular distribution it can be postulated that HtrA1 acts on different targets, such as intracellular growth factors or extracellular matrix proteins, to favor the correct formation/function of the placenta.

The ultrastructure of Makinoella tosaensis OKADA (Chlorophyta, Oocystaceae)

The cell wall of Makinoella tosaensis comprises two layers with relatively thin, parallel cellulose microfibrils which are arranged crosswise. A unique feature is a large extracytoplasmic space between plasma membrane and cell wall. It is filled with amorphous material, presumably of acidic polysaccharides and exocytosed by GOLGI vesicles. The structure of the dictyosomes is best understood by the cistemal progression model. A special characteristic are the pyrenoids. They are surrounded by a single, bowl-like starch grain. A funnelshaped thylakoid extends through the opening of the bowl into the pyrenoid matrix. Opposite to the opening there is a group of microbodies and mitochondria. The functional meaning of this association is discussed. The ultrastructural details confirm the inclusion of Makinoella in the Oocystaceae.

Membrane orientation of carboxyl-terminal half P-glycoprotein: Topogenesis of transmembrane segments

Multiple topological orientations of the carboxyl-terminal half of P-glycoprotein have been observed. One orientation is consistent with the hydropathy-predicted model and contains six transmembrane (TM)-spanning regions. In another orientation, the cytoplasmic-predicted loop between TM8 and TM9 is extracellular and glycosylated. In support of this “alternative” topology, TM8 was previously established to function as a signal-anchor sequence to insert with its amino-terminal end in the cytoplasm and the carboxyl-terminal end in the extracytoplasmic space. However, it is unclear how downstream TM segments fold in the membrane when TM8 functions as a signal-anchor sequence. Here, we created several chimeric Pgp molecules to examine the membrane insertion of TM segments 9 and 10 using a cell-free system. We found that TM9 functions as a stop-transfer sequence when following the signal-anchor sequence, T8S. However, the stop-transfer activity of TM9 depends on the presence of TM1O. In the absence of TM1O, TM9 partially translocated across the membrane into the endoplasmic reticulum lumen. In contrast, TM9 efficiently stopped the translocation event of the nascent chain in the presence of TM1O. Our results suggest that the membrane insertion of TM8 and TM9 establishes the extracellular loop between TM8 and TM9. Formation of this loop apparently involves the interactions between Pgp TM segments, which facilitate proper folding of the Pgp carboxyl-terminal half.

Sirohaem sulfite reductase and other proteins encoded by genes at the dsr locus of Chromatium vinosum are involved in the oxidation of intracellular sulfur.

The sequence of the dsr gene region of the phototrophic sulfur bacterium Chromatium vinosum D (DSMZ 180) was determined to clarify the in vivo role of 'reverse' sirohaem sulfite reductase. The dsrAB genes encoding dissimilatory sulfite reductase are part of a gene cluster, dsrABEFHCMK, that encodes four small, soluble proteins (DsrE, DsrF, DsrH and DsrC), a transmembrane protein (DsrM) with similarity to haem-b-binding polypeptides and a soluble protein (DsrK) resembling [4Fe-4S]-cluster-containing heterodisulfide reductase from methanogenic archaea. Northern hybridizations showed that expression of the dsr genes is increased by the presence of reduced sulfur compounds. The dsr genes are not only transcribed from a putative promoter upstream of dsrA but primary transcripts originating from (a) transcription start site(s) downstream of dsrB are also formed. Polar insertion mutations immediately upstream of dsrA, and in dsrB, dsrH and dsrM, led to an inability of the cells to oxidize intracellularly stored sulfur. The capability of the mutants to oxidize sulfide, thiosulfate and sulfite under photolithoautotrophic conditions was unaltered. Photoorganoheterotrophic growth was also unaffected. 'Reverse' sulfite reductase and DsrEFHCMK are, therefore, not essential for oxidation of sulfide or thiosulfate, but are obligatory for sulfur oxidation. These results, together with the finding that the sulfur globules of C. vinosum are located in the extracytoplasmic space whilst the dsr gene products appear to be either cytoplasmic or membrane-bound led to the proposal of new models for the pathway of sulfur oxidation in this phototrophic sulfur bacterium.

Expression of a synthetic antifreeze protein in potato reduces electrolyte release at freezing temperatures.

A synthetic antifreeze protein gene was expressed in plants and reduced electrolyte leakage from the leaves at freezing temperatures. The synthetic AFP was expressed as a fusion to a signal peptide, directing it to the extracytoplasmic space where ice crystallization first occurs. The gene was introduced to Solanum tuberosum L. cv. Russet Burbank by Agrobacterium-mediated transformation. Transformants were identified by PCR screening and expression of the introduced protein was verified by immunoblot. Electrolyte-release analysis of transgenic plant leaves established a correlation between the level of transgenic protein expression and degree of tolerance to freezing. This is the first identification of a phenotype associated with antifreeze protein expression in plant tissue.