Intracellular Accumulation of High Levels of γ-Aminobutyrate by Listeria monocytogenes 10403S in Response to Low pH: Uncoupling of γ-Aminobutyrate Synthesis from Efflux in a Chemically Defined Medium

It is well established that the glutamate decarboxylase (GAD) system is central to the survival of Listeria monocytogenes at low pH, both in acidic foods and within the mammalian stomach. The accepted model proposes that under acidic conditions extracellular glutamate is transported into the cell in exchange for an intracellular γ-aminobutyrate (GABA_i). The glutamate is then decarboxylated to GABA_i, a reaction that consumes a proton, thereby helping to prevent acidification of the cytoplasm. In this study, we show that glutamate supplementation had no influence on either growth rate at pH 5.0 or survival at pH 2.5 when L. monocytogenes 10403S was grown in a chemically defined medium (DM). In response to acidification, cells grown in DM failed to efflux GABA, even when glutamate was added to the medium. In contrast, in brain heart infusion (BHI), the same strain produced significant extracellular GABA (GABA_e) in response to acidification. In addition, high levels of GABA_i (>80 mM) were found in the cytoplasm in response to low pH in both growth media. Medium-swap and medium-mixing experiments revealed that the GABA efflux apparatus was nonfunctional in DM, even when glutamate was present. It was also found that the GadT2D2 antiporter/decarboxylase system was transcribed poorly in DM-grown cultures while overexpression of gadD1T1 and gadD3 occurred in response to pH 3.5. Interestingly, BHI-grown cells did not respond with upregulation of any of the GAD system genes when challenged at pH 3.5. The accumulation of GABA_i in cells grown in DM in the absence of extracellular glutamate indicates that intracellular glutamate is the source of the GABA_i. These results demonstrate that GABA production can be uncoupled from GABA efflux, a finding that alters the way we should view the operation of bacterial GAD systems.The capacity to produce γ-aminobutyric acid (GABA) through glutamate decarboxylation is commonly found in both Gram-negative and Gram-positive bacterial genera (10, 12). In several cases, this reaction has been shown to be critical for bacteria to survive potentially lethal acidic environments (15, 18, 20). It is generally held that the hydrogen ion consumed during the decarboxylation reaction helps to prevent excessive acidification of the cytoplasm, thereby protecting the cells against acidic environments. The GABA produced in the reaction is removed from the cell through the activity of an antiporter that exchanges a GABA molecule for an extracellular glutamate (Glu) molecule (6, 12).In Listeria monocytogenes, the Gram-positive food-borne pathogen that was the focus of the present study, the glutamate decarboxylase (GAD) system has been shown to play an essential role in acid tolerance (8, 9). Mutants compromised in their ability to catalyze this decarboxylation reaction survive poorly both in acidic foods (8) and gastric juice (9). The GAD system in most L. monocytogenes strains is encoded by a total of five genes. There are three genes, designated gadD1, gadD2, and gadD3, that encode distinct glutamate decarboxylase enzymes and two genes, designated gadT1 and gadT2, that encode two Glu-GABA antiporters. These genes are organized at three separate genetic loci: gadD1T1, gadT2D2, and gadD3 (11). The decarboxylase/antiporter system encoded by gadT2D2 plays a central role in allowing survival under extreme acidic conditions; mutants lacking either the GadT2 antiporter or the GadD2 decarboxylase are highly sensitive to low pH (9, 11). In contrast, the GadD1/GadT1 decarboxylase/antiporter system appears to be more important for growth under moderately acidic conditions (11). The genes encoding this system are absent from most serotype 4 strains, and this generally correlates with a reduced ability of these strains to grow well at low pH (11). The role of GadD3 is less clear since it has not been possible to generate a deletion mutant lacking the corresponding gene (9).Although the activity of the decarboxylase is generally thought to be coupled directly to the antiporter activity (i.e., the efflux of GABA is coupled to the supply of Glu) there is little direct evidence for this, even in bacteria where the system has been very well characterized. Most studies of the bacterial GAD system have used complex growth media when studying acid tolerance and GABA production (7, 8, 15). In the present study, we sought to determine whether extracellular Glu is a requirement for the production of GABA in L. monocytogenes. To do this, we have used a chemically defined growth medium (DM) that supports the growth of L. monocytogenes but does not include Glu. The results indicate that cells cultured in this medium do not produce extracellular GABA (GABA_e) in response to low pH but are capable of accumulating substantial pools of intracellular GABA (GABA_i). We establish that some component of complex medium is indispensable for efficient efflux of GABA. Surprisingly, supplementation of the DM with Glu failed to stimulate the extracellular release of GABA. We show that the GadD2/GadT2 decarboxylase/antiporter system is not transcribed when cells are grown in DM and suggest that this accounts for much of the difference in GABA production between cells cultured in DM and complex growth medium.     

Effect of colony morphology variation of <Emphasis Type="Italic">Burkholderia pseudomallei</Emphasis> on intracellular survival and resistance to antimicrobial environments in human macrophages in vitro

Background  

Primary diagnostic cultures from patients with melioidosis demonstrate variation in colony morphology of the causative organism, Burkholderia pseudomallei. Variable morphology is associated with changes in the expression of a range of putative virulence factors. This study investigated the effect of B. pseudomallei colony variation on survival in the human macrophage cell line U937 and under laboratory conditions simulating conditions within the macrophage milieu. Isogenic colony morphology types II and III were generated from 5 parental type I B. pseudomallei isolates using nutritional limitation. Survival of types II and III were compared with type I for all assays.     

A framework for power and sensitivity analyses for quantitative genetic studies of natural populations, and case studies in Soay sheep (Ovis aries)

Studies of the quantitative genetics of natural populations have contributed greatly to evolutionary biology in recent years. However, while pedigree data required are often uncertain (i.e. incomplete and partly erroneous) and limited, means to evaluate the effects of such uncertainties have not been developed. We have therefore developed a general framework for power and sensitivity analyses of such studies. We propose that researchers first generate a set of pedigree data that they wish to use in a quantitative genetic study, as well as data regarding errors that occur in that pedigree. This pedigree is then permuted using the data regarding errors to generate hypothetical 'true' and 'assumed' pedigrees that differ so as to mimic pedigree errors that might occur in the study system under consideration. Phenotypic data are then simulated across the true pedigree (according to user-defined genetic and environmental covariance structures), before being analysed with standard quantitative genetic techniques in conjunction with the 'assumed' pedigree data. To illustrate this approach, we conducted power and sensitivity analyses in a well-known study of Soay sheep (Ovis aries). We found that, although the estimation of simple genetic (co)variance structures is fairly robust to pedigree errors, some potentially serious biases were detected under more complex scenarios involving maternal effects. Power analyses also showed that this study system provides high power to detect heritabilities as low as about 0.09. Given this range of results, we suggest that such power and sensitivity analyses could greatly complement empirical studies, and we provide the computer program PEDANTICS to aid in their application.     

Morphological variation in the electric organ of the little skate (Leucoraja erinacea) and its possible role in communication during courtship

Skates discharge an electrical current too weak to be used for predation or defense, and too infrequent and irregular to be used for electrolocation. Additionally, skates possess a specialized sensory system that can detect electrical stimuli at the same strength at which they discharge their organs. These two factors are suggestive of a communicative role for the electric organ in skates, a role that has been demonstrated in similarly weakly electric teleosts (e.g., mormyrids and gymnotiforms). There is evidence that the sexual and ontogenetic variations in the electric organ discharge (EOD) in these other weakly electric fishes are linked to morphological variations in electric organs and the electrogenerating cells of the organs, the electrocytes. Little work has been done to examine possible sexual and ontogenetic variations in skate EODs or variations in the electrocytes responsible for those discharges. Electric organs and electrocyte morphology of male and female, and mature and immature little skates, Leucoraja erinacea, are characterized here. Female electric organs were bigger than male electric organs. This is suggestive of a sexually dimorphic EOD waveform or amplitude, which might be used as a sex-specific identification signal during courtship. The shapes of electrocytes that make up the organ were found to be significantly different between mature and immature individuals and, in some cases, posterior membrane surface area of the electrocytes increased at the onset of maturity due to the formation of membrane surface invaginations and papillae. This is evidence that the EOD of skates may differ in its waveform or amplitude or frequency between mature and immature skates, and act as a signal for readiness to mate. This study supports a communicative role during courtship for the weak electric organs of little skates, but studies that characterize skate EOD dimorphisms are needed to corroborate this speculation before conclusions can be drawn about the role the electric organ plays in communication during courtship.     

<Emphasis Type="Italic">Burkholderia</Emphasis> Hep_Hag autotransporter (BuHA) proteins elicit a strong antibody response during experimental glanders but not human melioidosis

Background  

The bacterial biothreat agents Burkholderia mallei and Burkholderia pseudomallei are the cause of glanders and melioidosis, respectively. Genomic and epidemiological studies have shown that B. mallei is a recently emerged, host restricted clone of B. pseudomallei.     

The genetic architecture of sexually selected traits in two natural populations of Drosophila montana

We investigated the genetic architecture of courtship song and cuticular hydrocarbon traits in two phygenetically distinct populations of Drosophila montana. To study natural variation in these two important traits, we analysed within-population crosses among individuals sampled from the wild. Hence, the genetic variation analysed should represent that available for natural and sexual selection to act upon. In contrast to previous between-population crosses in this species, no major quantitative trait loci (QTLs) were detected, perhaps because the between-population QTLs were due to fixed differences between the populations. Partitioning the trait variation to chromosomes suggested a broadly polygenic genetic architecture of within-population variation, although some chromosomes explained more variation in one population compared with the other. Studies of natural variation provide an important contrast to crosses between species or divergent lines, but our analysis highlights recent concerns that segregating variation within populations for important quantitative ecological traits may largely consist of small effect alleles, difficult to detect with studies of moderate power.     

Resource effects on denitrification are mediated by community composition in tidal freshwater wetlands soils

Accurate prediction of denitrification rates remains difficult, potentially owing to complex uncharacterized interactions between resource conditions and denitrifier communities. To better understand how the availability of organic matter (OM) and nitrate (NO₃^–), two of the resources most fundamental to denitrifiers, affect these populations and their activity, we performed an in situ resource manipulation in tidal freshwater wetland soils. Soils were augmented with OM to double ambient concentrations, using either compost or plant litter, and fertilized with KNO₃ at two levels (low: ～ 5 mg l^–1 NO₃^––N and high: ～ 50 mg l^–1 NO₃^––N) in a full factorial design. Community composition of nirS‐denitrifers (assessed using terminal restriction fragment length polymorphism) was interactively regulated by both NO₃^– concentration and OM type, and the associated shifts in community composition were relatively consistent across sampling dates (6, 9 and 12 months of incubation). Denitrification potential (pDNF) rates were also strongly affected by NO₃^– fertilization and increased by ～ 10–100‐fold. Path analysis revealed that the influence of resource availability on pDNF rates was largely mediated through changes in nirS‐denitrifier community composition. These results suggest that a greater understanding of denitrifier community ecology may enable more accurate prediction of denitrification rates.     

Bronchoabsorption; a novel bronchoscopic technique to improve biomarker sampling of the airway

Background

Current techniques used to obtain lung samples have significant limitations and do not provide reproducible biomarkers of inflammation. We have developed a novel technique that allows multiple sampling methods from the same area (or multiple areas) of the lung under direct bronchoscopic vision. It allows collection of mucosal lining fluid and bronchial brushing from the same site; biopsy samples may also be taken. The novel technique takes the same time as standard procedures and can be conducted safely.

Methods

Eight healthy smokers aged 40–65 years were included in this study. An absorptive filter paper was applied to the bronchial mucosa under direct vision using standard bronchoscopic techniques. Further samples were obtained from the same site using bronchial brushings. Bronchoalveolar lavage (BAL) was obtained using standard techniques. Chemokine (C-C Motif) Ligand 20 (CCL20), CCL4, CCL5, Chemokine (C-X-C Motif) Ligand 1 (CXCL1), CXCL8, CXCL9, CXCL10, CXCL11, Interleukin 1 beta (IL-1β), IL-6, Vascular endothelial growth factor (VEGF), Matrix metalloproteinase 8 (MMP-8) and MMP-9 were measured in exudate and BAL. mRNA was collected from the bronchial brushings for gene expression analysis.

Results

A greater than 10 fold concentration of all the biomarkers was detected in lung exudate in comparison to BAL. High yield of good quality RNA with RNA integrity numbers (RIN) between 7.6 and 9.3 were extracted from the bronchial brushings. The subset of genes measured were reproducible across the samples and corresponded to the inflammatory markers measured in exudate and BAL.

Conclusions

The bronchoabsorption technique as described offers the ability to sample lung fluid direct from the site of interest without the dilution effects caused by BAL. Using this method we were able to successfully measure the concentrations of biomarkers present in the lungs as well as collect high yield mRNA samples for gene expression analysis from the same site. This technique demonstrates superior sensitivity to standard BAL for the measurement of biomarkers of inflammation. It could replace BAL as the method of choice for these measurements. This method provides a systems biology approach to studying the inflammatory markers of respiratory disease progression.

Trial registration

NHS Health Research Authority (13/LO/0256).     

Basement membrane sliding and targeted adhesion remodels tissue boundaries during uterine-vulval attachment in Caenorhabditis elegans

Large gaps in basement membrane occur at sites of cell invasion and tissue remodelling in development and cancer. Though never followed directly in vivo, basement membrane dissolution or reduced synthesis have been postulated to create these gaps. Using landmark photobleaching and optical highlighting of laminin and type IV collagen, we find that a new mechanism, basement membrane sliding, underlies basement membrane gap enlargement during uterine-vulval attachment in Caenorhabditis elegans. Laser ablation and mutant analysis reveal that the invaginating vulval cells promote basement membrane movement. Further, an RNA interference and expression screen identifies the integrin INA-1/PAT-3 and VAB-19, homologue of the tumour suppressor Kank, as regulators of basement membrane opening. Both concentrate within vulval cells at the basement membrane gap boundary and halt expansion of the shifting basement membrane. Basement membrane sliding followed by targeted adhesion represents a new mechanism for creating precise basement membrane breaches that can be used by cells to break down compartment boundaries.