High-osmolarity Medium Research Articles

Cloning and characterization of a NAD+-dependent glycerol-3-phosphate dehydrogenase gene fromCandida glycerinogenes, an industrial glycerol producer

The osmotolerant yeast Candida glycerinogenes produces glycerol as a major metabolite on an industrial scale, but the underlying molecular mechanisms are poorly understood. We cloned and characterized a 4900-bp genomic fragment containing the CgGPD gene encoding a glycerol-3-phosphate dehydrogenase homologous to GPD genes in other yeasts using degenerate primers in conjunction with inverse PCR. Sequence analysis revealed a 1167-bp open reading frame encoding a putative peptide of 388 deduced amino acids with a molecular mass of 42 695 Da. The CgGPD gene consisted of an N-terminal NAD(+)-binding domain and a central catalytic domain, whereas seven stress response elements were found in the upstream region. Functional analysis revealed that Saccharomyces cerevisiae gpd1Delta and gpd1Delta/gpd2Delta osmosensitive mutants transformed with CgGPD were restored to the wild-type phenotype when cultured in high osmolarity media, suggesting that it is a functional GPD protein. Transformants also accumulated glycerol intracellularly and GPD-specific activity increased significantly when stressed with NaCl, whereas the S. cerevisiae mutants transformed with the empty plasmid showed only slight increases. The full-length CgGPD gene sequence including upstream and downstream regions has been deposited in GenBank under accession no. EU186536.

Genome-Wide Transcriptional Responses ofEscherichia coliK-12 to Continuous Osmotic and Heat Stresses

Osmotic stress is known to increase the thermotolerance and oxidative-stress resistance of bacteria by a mechanism that is not adequately understood. We probed the cross-regulation of continuous osmotic and heat stress responses by characterizing the effects of external osmolarity (0.3 M versus 0.0 M NaCl) and temperature (43 degrees C versus 30 degrees C) on the transcriptome of Escherichia coli K-12. Our most important discovery was that a number of genes in the SoxRS and OxyR oxidative-stress regulons were up-regulated by high osmolarity, high temperature, or a combination of both stresses. This result can explain the previously noted cross-protection of osmotic stress against oxidative and heat stresses. Most of the genes shown in previous studies to be induced during the early phase of adaptation to hyperosmotic shock were found to be also overexpressed under continuous osmotic stress. However, there was a poorer overlap between the heat shock genes that are induced transiently after high temperature shifts and the genes that we found to be chronically up-regulated at 43 degrees C. Supplementation of the high-osmolarity medium with the osmoprotectant glycine betaine, which reduces the cytoplasmic K(+) pool, did not lead to a universal reduction in the expression of osmotically induced genes. This finding does not support the hypothesis that K(+) is the central osmoregulatory signal in Enterobacteriaceae.

Metabolic activity of Siberian permafrost isolates, Psychrobacter arcticus and Exiguobacterium sibiricum, at low water activities

The Siberian permafrost is an extreme, yet stable environment due to its continuously frozen state. Microbes maintain membrane potential and respiratory activity at average temperatures of -10 to -12 degrees C that concentrate solutes to an aw=0.90 (5 osm), The isolation of viable Psychrobacter arcticus sp. 273-4 and Exiguobacterium sibiricum sp. 255-15 from ancient permafrost suggests that these bacteria have maintained some level of metabolic activity for thousands of years. Permafrost water activity was simulated using 1/2 TSB+2.79 m NaCl (5 osm) at and cells were held at 22 and 4 degrees C. Many cells reduced cyano-tetrazolium chloride (CTC) indicating functioning electron transport systems. Increased membrane permeability was not responsible for this lack of electron transport, as more cells were determined to be intact by LIVE/DEAD staining than were reducing CTC. Low rates of aerobic respiration were determined by the slope of the reduced resazurin line for P. arcticus, and E. sibiricum. Tritiated leucine was incorporated into new proteins at rates indicating basal level metabolism. The continued membrane potential, electron transport and aerobic respiration, coupled with incorporation of radio-labeled leucine into cell material when incubated in high osmolarity media, show that some of the population is metabolically active under simulated in situ conditions.

Responses of Bacillus subtilis to Hypotonic Challenges: Physiological Contributions of Mechanosensitive Channels to Cellular Survival

Mechanosensitive channels are thought to function as safety valves for the release of cytoplasmic solutes from cells that have to manage a rapid transition from high- to low-osmolarity environments. Subsequent to an osmotic down-shock of cells grown at high osmolarity, Bacillus subtilis rapidly releases the previously accumulated compatible solute glycine betaine in accordance with the degree of the osmotic downshift. Database searches suggest that B. subtilis possesses one copy of a gene for a mechanosensitive channel of large conductance (mscL) and three copies of genes encoding proteins that putatively form mechanosensitive channels of small conductance (yhdY, yfkC, and ykuT). Detailed mutational analysis of all potential channel-forming genes revealed that a quadruple mutant (mscL yhdY yfkC ykuT) has no growth disadvantage in high-osmolarity media in comparison to the wild type. Osmotic down-shock experiments demonstrated that the MscL channel is the principal solute release system of B. subtilis, and strains with a gene disruption in mscL exhibited a severe survival defect upon an osmotic down-shock. We also detected a minor contribution of the SigB-controlled putative MscS-type channel-forming protein YkuT to cellular survival in an mscL mutant. Taken together, our data revealed that mechanosensitive channels of both the MscL and MscS types play pivotal roles in managing the transition of B. subtilis from hyper- to hypo-osmotic environments.

Role of outer membrane lipopolysaccharides in the protection of Salmonella enterica serovar Typhimurium from desiccation damage

The ability to survive desiccation between hosts is often essential to the success of pathogenic bacteria. The bacterial outer membrane is both the cellular interface with hostile environments and the focus of much of the drying-induced damage. This study examined the contribution of outer membrane-associated polysaccharides to the survival of Salmonella enterica serovar Typhimurium in air-dried blood droplets following growth in high and low osmolarity medium and under conditions known to induce expression of these polysaccharides. Strains lacking the O polysaccharide (OPS) element of the outer membrane lipopolysaccharide were more sensitive to desiccation. Lipopolysaccharide core mutation further to OPS loss did not result in increased susceptibility to drying. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed lipopolysaccharide profiles that supported the hypothesis that OPS expression is required for optimal drying resistance in S. Typhimurium. The role of O antigen in Salmonella spp. in maintaining a hydrated layer around the dried cell or in slowing the rate of dehydration and rehydration is discussed.

Novel Mitogen-Activated Protein Kinase MpkC of Aspergillus fumigatus Is Required for Utilization of Polyalcohol Sugars

The genome of Aspergillus fumigatus has four genes that encode mitogen-activated protein kinases (MAPKs), sakA/hogA, mpkA, mpkB, and mpkC. The functions of the MpkB and MpkC MAPKs are unknown for A. fumigatus and the closely related and genetically amenable species Aspergillus nidulans. mpkC deletion mutants of A. fumigatus were made and their phenotypes characterized. The mpkC deletion mutants were viable and had normal conidial germination and hyphal growth on minimal or complete media. This is in contrast to deletion mutants with deletions in the closely related MAPK gene sakA/hogA that we previously reported had a nitrogen source-dependent germination phenotype. Similarly, the growth of the mpkC deletion mutants was wild type on high-osmolarity medium. Consistent with these two MAP kinase genes regulating different cellular responses, we determined that the mpkC deletion mutants were unable to grow on minimal medium with sorbitol or mannitol as the sole carbon source. This result implicates MpkC signaling in carbon source utilization. Changes in mRNA levels for sakA and mpkC were measured in response to hypertonic stress, oxidative stress, and a shift from glucose to sorbitol to determine if there was overlap in the SakA and MpkC signaling pathways. These studies demonstrated that SakA- and MpkC-dependent patterns of change in mRNA levels are distinct and have minimal overlap in response to these environmental stresses.

371 INTRACYTOPLASMIC SPERM INJECTION OF EQUINE OOCYTES USING AIR-DRIED SPERM OR SPERM STORED IN A HIGH OSMOLARITY MEDIUM

The generally accepted method of long-term sperm preservation is freezing in liquid nitrogen. However, it is not always available. Other techniques have shown to preserve sperm for a short period of time that can be used for intracytoplasmic sperm injection (ICSI). Mouse offspring have been produced after ICSI with sperm stored in a high osmolarity medium. Also, human embryos were obtained by ICSI with air-dried sperm. Recently, equine blastocysts have been obtained by ICSI with lyophilized sperm. In our study, cleavage rate was evaluated after ICSI of equine oocytes using air-dried sperm or sperm stored in a high osmolarity medium. Oocytes were obtained from slaughterhouse ovaries by scraping individual follicles and transported in a portable incubator at 38�C for 15 h in TCM-199 buffered with HEPES and supplemented with glutamine, sodium pyruvate, LH (Bioniche Animal Health, Inc., Beltville, Ontario, Canada), FSH (Bioniche Animal Health, Inc.), epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1), and 10% FBS (GIBCO, Grand Island, NY, USA). On arrival, they were cultured for 6 more hours in 5% CO2 in air at 38�C in microdroplets of the same medium, without HEPES. Sperm was collected from 2 stallions of proven fertility. The ejaculate was diluted in Kenney extender, centrifuged, and the pellet was resuspended in HEPES-TALP (H-TALP). Three sperm treatments were used: (C) Control: ejaculated motile sperm processed as described above; (1) Air-dried sperm: obtained from spreading sperm on a sterile slide and drying it for 10 min in a laminar flow chamber; (2) Sperm in a high osmolarity medium: ejaculated motile sperm resuspended in H-TALP with high osmolarity (800 mOsmol). Samples from groups 1 and 2 were stored at 5�C for 2 to 3 days before being used for sperm injection. Only oocytes with an intact cytoplasm and a visible polar body were selected for injection and randomly assigned to each experimental group. Each MII oocyte was injected with 1 sperm cell and activated in Ionomicin for 10 min and DMAP for 3 h. Injected oocytes were cultured in DMEM : F10 1 : 1 with 10% FBS at 38�C in 7% O2 and 5% CO2 for 48 h. The number of cleaved embryos was recorded. Data was analyzed by chi-square test. A total of 135 MII oocytes were injected. The cleavage rate in group 1 was significantly lower than in the control group (31/71, 43.66% vs. 28/38, 73.68%) (P < 0.05). No differences were observed between group (2) and control (19/29, 73.07% vs. 28/38, 73.68%) (P > 0.05). This is the first report of equine oocytes fertilized by ICSI with air-dried sperm or with sperm kept in high osmolarity medium. These simple sperm preservation techniques might be an alternative option when liquid nitrogen is not available. Further studies will determine if it is possible to obtain pregnancies or even healthy offspring.

The Sho1 Adaptor Protein Links Oxidative Stress to Morphogenesis and Cell Wall Biosynthesis in the Fungal Pathogen Candida albicans

The Sho1 adaptor protein is an important element of one of the two upstream branches of the high-osmolarity glycerol (HOG) mitogen-activated protein (MAP) kinase pathway in Saccharomyces cerevisiae, a signal transduction cascade involved in adaptation to stress. In the present work, we describe its role in the pathogenic yeast Candida albicans by the construction of mutants altered in this gene. We report here that sho1 mutants are sensitive to oxidative stress but that Sho1 has a minor role in the transmission of the phosphorylation signal to the Hog1 MAP kinase in response to oxidative stress, which mainly occurs through a putative Sln1-Ssk1 branch of the HOG pathway. Genetic analysis revealed that double ssk1 sho1 mutants were still able to grow on high-osmolarity media and activate Hog1 in response to this stress, indicating the existence of alternative inputs of the pathway. We also demonstrate that the Cek1 MAP kinase is constitutively active in hog1 and ssk1 mutants, a phenotypic trait that correlates with their resistance to the cell wall inhibitor Congo red, and that Sho1 is essential for the activation of the Cek1 MAP kinase under different conditions that require active cell growth and/or cell wall remodeling, such as the resumption of growth upon exit from the stationary phase. sho1 mutants are also sensitive to certain cell wall interfering compounds (Congo red, calcofluor white), presenting an altered cell wall structure (as shown by the ability to aggregate), and are defective in morphogenesis on different media, such as SLAD and Spider, that stimulate hyphal growth. These results reveal a role for the Sho1 protein in linking oxidative stress, cell wall biogenesis, and morphogenesis in this important human fungal pathogen.

Role for Glycine Betaine Transport in Vibrio cholerae Osmoadaptation and Biofilm Formation within Microbial Communities

Vibrio cholerae is a halophilic facultative human pathogen found in marine and estuarine environments. Accumulation of compatible solutes is important for growth of V. cholerae at NaCl concentrations greater than 250 mM. We have identified and characterized two compatible solute transporters, OpuD and PutP, that are involved in uptake of glycine betaine and proline by V. cholerae. V. cholerae does not, however, possess the bet genes, suggesting that it is unable to synthesize glycine betaine. In contrast, many Vibrio species are able to synthesize glycine betaine from choline. It has been shown that many bacteria not only synthesize but also secrete glycine betaine. We hypothesized that sharing of compatible solutes might be a mechanism for cooperativity in microbial communities. In fact, we have demonstrated that, in high-osmolarity medium, V. cholerae growth and biofilm development are enhanced by supplementation with either glycine betaine or spent media from other bacterial species. Thus, we propose that compatible solutes provided by other microorganisms may contribute to survival of V. cholerae in the marine environment through facilitation of osmoadaptation and biofilm development.

CpxR/OmpR interplay regulates curli gene expression in response to osmolarity in Escherichia coli.

Curli fibers could be described as a virulence factor able to confer adherence properties to both abiotic and eukaryotic surfaces. The ability to adapt rapidly to changing environmental conditions through signal transduction pathways is crucial for the growth and pathogenicity of bacteria. OmpR was shown to activate csgD expression, resulting in curli production. The CpxR regulator was shown to negatively affect curli gene expression when binding to its recognition site that overlaps the csgD OmpR-binding site. This study was undertaken to clarify how the interplay between the two regulatory proteins, OmpR and CpxR, can affect the transcription of the curli gene in response to variation of the medium osmolarity. Band-shift assays with purified CpxR proteins indicate that CpxR binds to the csgD promoter region at multiple sites that are ideally positioned to explain the csg repression activity of CpxR. To understand the physiological meaning of this in vitro molecular phenomenon, we analyzed the effects of an osmolarity shift on the two-component pathway CpxA/CpxR. We establish here that the Cpx pathway is activated at both transcriptional and posttranscriptional levels in response to a high osmolarity medium and that CpxR represses csgD expression in high-salt-content medium, resulting in low curli production. However, csgD repression in response to high sucrose content is not mediated by CpxR but by the global regulatory protein H-NS. Therefore, multiple systems (EnvZ/OmpR, Cpx, Rcs, and H-NS) appear to be involved in sensing environmental osmolarity, leading to sophisticated regulation of the curli genes.

Lactococcus lactis Uses MscL as Its Principal Mechanosensitive Channel

The functions of the mechanosensitive channels from Lactococcus lactis were determined by biochemical, physiological, and electrophysiological methods. Patch-clamp studies showed that the genes yncB and mscL encode MscS and MscL-like channels, respectively, when expressed in Escherichia coli or if the gene products were purified and reconstituted in proteoliposomes. However, unless yncB was expressed in trans, wild type membranes of L. lactis displayed only MscL activity. Membranes prepared from an mscL disruption mutant did not show any mechanosensitive channel activity, irrespective of whether the cells had been grown on low or high osmolarity medium. In osmotic downshift assays, wild type cells survived and retained 20% of the glycine betaine internalized under external high salt conditions. On the other hand, the mscL disruption mutant retained 40% of internalized glycine betaine and was significantly compromised in its survival upon osmotic downshifts. The data strongly suggest that L. lactis uses MscL as the main mechanosensitive solute release system to protect the cells under conditions of osmotic downshift.

A simulation model of Escherichia coli osmoregulatory switch using E-CELL system

BackgroundBacterial signal transduction mechanism referred to as a "two component regulatory systems" contributes to the overall adaptability of the bacteria by regulating the gene expression. Osmoregulation is one of the well-studied two component regulatory systems comprising of the sensor, EnvZ and the cognate response regulator, OmpR, which together control the expression of OmpC and OmpF porins in response to the osmolyte concentration.ResultsA quantitative model of the osmoregulatory switch operative in Escherichia coli was constructed by integrating the enzyme rate equations using E-CELL system. Using the substance reactor logic of the E-CELL system, a total of 28 reactions were defined from the injection of osmolyte till the regulated expression of porins by employing the experimental kinetic constants as reported in literature. In the case of low osmolarity, steady state production of OmpF and repression of OmpC was significant. In this model we show that the steady state – production of OmpF is dramatically reduced in the high osmolarity medium. The rate of OmpC production increased after sucrose addition, which is comparable with literature results. The relative porin production seems to be unaltered with changes in cell volume changes, ATP, EnvZ and OmpR at low and high osmolarity conditions. But the reach of saturation was rapid at high and low osmolarity with altered levels of the above components.ConclusionsThe E-CELL system allows us to perform virtual experiments on the bacterial osmoregulation model. This model does not take into account interaction with other networks in the cell. It suggests that the regulation of OmpF and OmpC is a direct consequence of the level of OmpRP in the cell and is dependent on the way in which OmpRP interacts with ompF and ompC regulatory regions. The preliminary simulation experiment indicates that both reaching steady state expression and saturation is delayed in the case of OmpC compared to OmpF. Experimental analysis will help improve the model. The model captures the basic features of the generally accepted view of EnvZ-OmpR signaling and is a reasonable starting point for building sophisticated models and explaining quantitative features of the system.

Role of ectoine in Vibrio cholerae osmoadaptation.

Vibrio cholerae is both an intestinal pathogen and a microbe in the estuarine community. To persist in the estuarine environment, V. cholerae must adjust to changes in ionic composition and osmolarity. These changes in the aquatic environment have been correlated with cholera epidemics. In this work, we study the response of V. cholerae to increases in environmental osmolarity. Optimal growth of V. cholerae in minimal medium requires supplementation with 200 mM NaCl and KCl. However, when the NaCl concentration is increased beyond 200 mM, a proportionate delay in growth is observed. During this delay in growth, osmotic equilibrium is reached by cytoplasmic accumulation of small, uncharged solutes that are compatible with growth. We show that synthesis of the compatible solute ectoine and transport of the compatible solute glycine betaine impact the length of the osmoadaptive growth delay. We also demonstrate that high-osmolarity-adapted V. cholerae displays a growth advantage when competed against unadapted cells in high-osmolarity medium. In contrast, low-osmolarity-adapted V. cholerae displays no growth advantage when competed against high-osmolarity-adapted cells in low-osmolarity medium. These results may have implications for V. cholerae population dynamics when seawater and freshwater and their attendant microbes mix.

Adaptation to high salt in Lactobacillus: role of peptides and proteolytic enzymes.

To study the influence of peptides and proteolytic enzymes in the osmotic adaptation of Lactobacillus casei. Di- and tri-peptides added individually increased the osmotolerance of Lact. casei when grown in a chemically defined medium (CDM) containing NaCl. Growth stimulation and the re-establishment in their presence of plasmid DNA supercoiling (recovery of the linking number) in hyperosmotic medium indicated that they are used as osmocompatible solutes as carnithine a known osmoprotector does. The investigation of the proteolytic system showed that in high osmolarity medium, the cell envelope-associated proteinase (PrtP), and PepX (X-prolyl-dipeptidyl aminopeptidase) increased activity and lost repression by peptides. PepI, an iminopeptidase was also derepressed. PepQ, a prolidase that specifically liberated proline from dipeptides, was almost unaffected. Derepression in the presence of peptides took place at the transcriptional level. However, the twofold activation of PrtP in CDM hyperosmotic medium was essentially through an increase of the apparent Vmax of the enzyme. These results strongly suggest a contribution of the proteolytic system peptide supply in the osmotic adaptation. Advances in understanding the role of peptides in the adaptation to high osmolarity particularly involved in dairy processes.

A genetic screen for yeast genes induced by sustained osmotic stress.

The budding yeast, Saccharomyces cerevisiae, responds to changes in external osmolarity through the activation of an osmosensing signal transduction pathway. Using lacZ-reporter gene fusions, clonal cell lines were screened for levels of beta-galactosidase activity in the presence or absence of osmotic stress. A screen of 9,000 transformants displayed 663 (7%) gene fusions that were active in rich medium. Each of the transformants were also assayed for gene activity 24 h following a transfer to high osmolarity medium (0.6 M NaCl) and of the 9,000 clonal cell lines, 86 (1%) displayed a decrease in expression, and seven (0.1%) displayed a reproducible increase in gene expression during primary screening. The chromosomal loci of the lacZ insertions were determined, and the gene(s) associated with that site was examined for osmotically induced expression using RNA blot analysis. Five stress-activated genes were analysed by RNA blot: YDL222C, NMD2, PTC7, FAA4 and YRF1. The genes identified by this screen encompass cellular adaptations to stress including signal transduction, protein myristoylation and fatty acid/sphingolipid content in the cell membrane.

Killing of Caenorhabditis elegans by Burkholderia cepacia is controlled by the cep quorum-sensing system.

Burkholderia cepacia H111, which was isolated from a cystic fibrosis patient, effectively kills the nematode Caenorhabditis elegans. Depending on the medium used for growth of the bacterium two different killing modes were observed. On high-osmolarity medium the nematodes became paralysed and died within 24 h. Using filter assays we provide evidence that this killing mode involves the production of an extracellular toxin. On nematode growth medium killing occurs over the course of 2-3 days and involves the accumulation of bacteria in the intestinal lumen of C. elegans. We demonstrate that the cep quorum-sensing system of H111 is required for efficient killing of C. elegans under both killing conditions. Using the C. elegans phm-2 mutant that has a non-functional grinder evidence is provided that the cep system is required to enter the intestinal lumen but is dispensable for the colonization of the gut. Furthermore, we demonstrate that the type II secretion machinery is not essential for nematode killing.

The phospholipase B homolog Plb1 is a mediator of osmotic stress response and of nutrient-dependent repression of sexual differentiation in the fission yeast Schizosaccharomyces pombe.

Although phospholipase B (PLB) enzymes have been described in eukaryotes from yeasts to mammals, their biological functions are poorly understood. Here we describe the characterization of plb1, one of five genes predicted to encode PLB homologs in the fission yeast, Schizosaccharomyces pombe. The plb1 gene is dispensable under normal growth conditions but required for viability in high-osmolarity media and for normal osmotic stress-induced gene expression. Unlike mutants defective in function for the stress-activated MAP kinase Spc1, plb1Delta cells are not hypersensitive to oxidative or temperature stresses, nor do they undergo a G2-specific arrest in response to osmotic stress. In addition to defects in osmotic stress response, plb1Delta cells exhibit a cold-sensitive defect in nutrient-mediated mating repression, a phenotype reminiscent of mutants in the cyclic AMP (cAMP) pathway. We show that, like plb1Delta cells, mutants in the cAMP pathway are defective for growth in high-osmolarity media, demonstrating a previously unrecognized role for the cAMP pathway in osmotic stress response. Furthermore, we show that gain-of function in the cAMP pathway can rescue the osmosensitive growth defect of plb1Delta cells, suggesting that the cAMP pathway is a potential downstream target of the actions of Plb1 in S. pombe.

Transient gene expression of beta-glucuronidase in citrus thin epicotyl transversal sections using particle bombardment

Studies were carried out to optimize the conditions for transient gene expression through particle bombardment on Carrizo citrange (Citrus sinensis x Poncirus trifoliata) thin epicotyl sections. The best conditions for transient GUS expression were: M-25 tungsten particles, 1550 psi helium pressure, 9 cm distance between specimen and DNA/particle holder and culture of explants in a high osmolarity medium (0.2 M mannitol + 0.2 M sorbitol) 4 h prior and 20 h after bombardment. Under these conditions, an average of 102 blue spots per bombardment (20 explants/plate) were achieved. This protocol is currently being used for transformation of Carrizo citrange and sweet orange (Citrus sinensis).

Characterization of a novel intracellularly activated gene from Salmonella enterica serovar typhi.

A Salmonella enterica serovar Typhi gene that is selectively up-regulated upon bacterial invasion of eukaryotic cells was characterized. The open reading frame encodes a 298-amino-acid hydrophobic polypeptide (30.8 kDa), which is predicted to be an integral membrane protein with nine membrane-spanning domains. The protein is closely related (87 to 94% reliability) to different transport and permease systems. Gene expression under laboratory conditions was relatively weak; however, sevenfold induction was observed in a high-osmolarity medium (300 mM NaCl). The growth pattern in a laboratory medium of a serovar Typhi strain Ty2 derivative containing a 735-bp in-frame deletion in this gene, named gaiA (for gene activated intracellularly), was not affected. In contrast, the mutant was partially impaired in intracellular survival in murine peritoneal macrophages, as well as in human monocyte-derived macrophages. However, in the case of human macrophages, this survival defect was modest and evident only at late infection times (24 h). Despite the distinct intracellular survival kinetics displayed in macrophages of different species, the gaiA null mutant was significantly affected in its potential to trigger apoptosis in both murine and human macrophages. Provision of the gaiA gene in trans resulted in complementation of these phenotypes. Interestingly, the absence of a functional gaiA gene caused a marked attenuation in the mouse mucin model, as shown by the increase (3 orders of magnitude) in the 50% lethal dose of the mutant strain over that of the parental strain Ty2 (P </= 0.05). Altogether, these data indicate that the product encoded by the gaiA gene is required for triggering apoptosis and bacterial survival within murine macrophages, which is consistent with the in vivo results obtained in the mouse mucin model. However, gaiA was not required for initial intracellular survival in human cells, indicating that its role in the natural host might be more complex than is suggested by the studies performed in the murine system.

Hydroxyectoine is superior to trehalose for anhydrobiotic engineering of Pseudomonas putida KT2440.

Anhydrobiotic engineering aims to increase the level of desiccation tolerance in sensitive organisms to that observed in true anhydrobiotes. In addition to a suitable extracellular drying excipient, a key factor for anhydrobiotic engineering of gram-negative enterobacteria seems to be the generation of high intracellular concentrations of the nonreducing disaccharide trehalose, which can be achieved by osmotic induction. In the soil bacterium Pseudomonas putida KT2440, however, only limited amounts of trehalose are naturally accumulated in defined high-osmolarity medium, correlating with relatively poor survival of desiccated cultures. Based on the enterobacterial model, it was proposed that increasing intracellular trehalose concentration in P. putida KT2440 should improve survival. Using genetic engineering techniques, intracellular trehalose concentrations were obtained which were similar to or greater than those in enterobacteria, but this did not translate into improved desiccation tolerance. Therefore, at least for some populations of microorganisms, trehalose does not appear to provide full protection against desiccation damage, even when present at high concentrations both inside and outside the cell. For P. putida KT2440, it was shown that this was not due to a natural limit in desiccation tolerance since successful anhydrobiotic engineering was achieved by use of a different drying excipient, hydroxyectoine, with osmotically preconditioned bacteria for which 40 to 60% viability was maintained over extended periods (up to 42 days) in the dry state. Hydroxyectoine therefore has considerable potential for the improvement of desiccation tolerance in sensitive microorganisms, particularly for those recalcitrant to trehalose.