A streptococcal NRAMP homologue is crucial for the survival of Streptococcus agalactiae under low pH conditions

Streptococcus agalactiae or Group B Streptococcus (GBS) is a commensal bacterium of the human gastrointestinal and urogenital tracts as well as a leading cause of neonatal sepsis, pneumonia and meningitis. Maternal vaginal carriage is the main source for GBS transmission and thus the most important risk factor for neonatal disease. Several studies in eukaryotes identified a group of proteins natural resistance‐associated macrophage protein (NRAMP) that function as divalent cation transporters for Fe²⁺ and Mn²⁺ and confer on macrophages the ability to control replication of bacterial pathogens. Genome sequencing predicted potential NRAMP homologues in several prokaryotes. Here we describe for the first time, a pH‐regulated NRAMP Mn²⁺/Fe²⁺ transporter in GBS, designated MntH, which confers resistance to reactive oxygen species (ROS) and is crucial for bacterial growth and survival under low pH conditions. Our investigation implicates MntH as an important colonization determinant for GBS in the maternal vagina as it helps bacteria to adapt to the harsh acidic environment, facilitates bacterial adherence, contributes to the coexistence with the vaginal microbiota and plays a role in GBS intracellular survival inside macrophages.     

Survival and biofilm formation by Group B streptococci in simulated vaginal fluid at different pHs

The Group B Streptococcus (GBS, Streptococcus agalactiae) is an important cause of neonatal and maternal infection. GBS is a commensal organism of the lower gastrointestinal and vaginal tract. A frequent mode of neonatal infection is vertical transmission from pregnant women to their foetus or neonate. The aim of this study was to evaluate the survival and biofilm production of 10 GBS strains in simulated vaginal fluid at pH 4.2, 5.5 and 6.5. GBS survived longer at higher pH than at normal vaginal pH. At pH 4.2, with the exception of two isolates that were recovered up to 48 and 72 h, viable GBS numbers declined below the limit of detection by 24 h. At higher pH, GBS survived between 3 and 15 days. All isolates investigated were biofilm producers but biofilm production was greater in tryptone soy broth compared to simulated vaginal fluid. The quantity of biofilm produced increased with the rise in the pH. This study suggests that high vaginal pH may influence both GBS survival and biofilm production and thus could be a risk factor for GBS infection.     

Expression of the MtsA lipoprotein of <Emphasis Type="Italic">Streptococcus agalactiae</Emphasis> A909 is regulated by manganese and iron

Metal ion acquisition and homeostasis are essential for bacterial survival, growth and physiology. A family of metal ion, ABC-type import systems have been identified in Gram-positive bacteria, in which the solute-binding proteins are predicted to be membrane-anchored lipoproteins. The prediction that the MtsA protein of Streptococcus agalactiae A909 is a lipoprotein was confirmed. The expression of MtsA was co-ordinately regulated by the presence of both manganese and ferrous ions suggesting that MtsA may be involved in the uptake of both these ions. MtsA was shown to be expressed at levels of ferrous ions known to be present in amniotic fluid, a growth medium for S. agalactiae during neonatal infection.     

Molecular typing of colonizing Streptococcus agalactiae strains by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) in a Chennai based hospital

Streptococcus agalactiae is reported to be an asymptomatic vaginal colonizer in Indian women, although it is considered one of the major causes of neonatal infections in many European countries. DNA based molecular typing methods are more reliable than the conventional serotyping method for identification and typing of this pathogen. In the present study, we have evaluated genetic diversity among colonizing S. agalactiae strains (n=86) by using a PCR-based genotyping method i.e. Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR). With ERIC-PCR fingerprinting at 60% similarity level in a dendrogram generated by UPGMA cluster analysis, 10 different ERIC groups were identified, which were subdivided into 62 distinct genotypes at ≥ 95% similarity level. Based on these findings, we demonstrate that ERIC-PCR is a simple, rapid, and inexpensive tool with sufficient discriminatory power and is applicable for characterization and genotyping of a large number of clinical isolates of S. agalactiae at molecular level.     

Testing the Stress‐Gradient Hypothesis During the Restoration of Tropical Degraded Land Using the Shrub Rhodomyrtus tomentosa as a Nurse Plant

The relative importance of facilitation and competition between pairwise plants across abiotic stress gradients as predicted by the stress‐gradient hypothesis has been confirmed in arid and temperate ecosystems, but the hypothesis has rarely been tested in tropical systems, particularly across nutrient gradients. The current research examines the interactions between a pioneer shrub Rhodomyrtus tomentosa (the nurse plant) and seedlings of a transplanted native woody Schima superba (the target species) in a tropical system in which position on a slope corresponds with a nutrient gradient; high soil nutrients at the slope bottom and relatively low soil nutrients at the slope top. In contrast, soil physical traits were more favorable for seedling growth under the shrub than in open spaces. The effect of R. tomentosa on S. superba survival was positive (facilitation) at the top of the slope, as indicated by the relative interaction index (RII), but negative in the bottom (competition). RII indicated a positive effect on seedling height at the top of the slope but was not at the bottom. Seedling survival was positively related to soil nutrient level and negatively related to soil acidity, but seedling growth of S. superba seemed to be enhanced by the shrub canopy. Thus, the results seem to support stress‐gradient hypothesis in terms of target species survival but not growth. We suggest using the shrub as a nurse plant in forest restoration in tropical degraded land with caution because not all of its effects on target species are positive .     

Survival and growth of two heterotrophic hydrothermal vent archaea, <Emphasis Type="Italic">Pyrococcus</Emphasis> strain GB-D and <Emphasis Type="Italic">Thermococcus fumicolans</Emphasis>, under low pH and high sulfide concentrations in combination with high temperature and pressure regimes

Growth and survival of hyperthermophilic archaea in their extreme hydrothermal vent and subsurface environments are controlled by chemical and physical key parameters. This study examined the effects of elevated sulfide concentrations, temperature, and acidic pH on growth and survival of two hydrothermal vent archaea (Pyrococcus strain GB-D and Thermococcus fumicolans) under high temperature and pressure regimes. These two strains are members of the Thermococcales, a family of hyperthermophilic, heterotrophic, sulfur-reducing archaea that occur in high densities at vent sites. As actively growing cells, these two strains tolerated regimes of pH, pressure, and temperature that were in most cases not tolerated under severe substrate limitation. A moderate pH of 5.5–7 extends their survival and growth range over a wider range of sulfide concentrations, temperature and pressure, relative to lower pH conditions. T. fumicolans and Pyrococcus strain GB-D grew under very high pressures that exceeded in-situ pressures typical of hydrothermal vent depths, and included deep subsurface pressures. However, under the same conditions, but in the absence of carbon substrates and electron acceptors, survival was generally lower, and decreased rapidly when low pH stress was combined with high pressure and high temperature. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Local Adaptation and Genetics of Acid-Stress Tolerance in the Moor Frog, Rana arvalis

As potential to adapt to environmental stress can be essential for population persistence, knowledge on the genetic architecture of local adaptation is important for conservation genetics. We investigated the relative importance of additive genetic, dominance and maternal effects contributions to acid stress tolerance in two moor frog (Rana arvalis) populations originating from low and neutral pH habitats. Experiments with crosses obtained from artificial matings revealed that embryos from the acid origin population were more tolerant to low pH than embryos from the neutral origin population in embryonic survival rates, but not in terms of developmental stability, developmental and growth rates. Strong maternal effect and small additive genetic contributions to variation were detected in all traits in both populations. In general, dominance contributions to variance in different traits were of similar magnitude to the additive genetic effects, but dominance effects outweighed the additive genetic and maternal effects contributions to early growth in both populations. Furthermore, the expression of additive genetic variance was independent of pH treatment, suggesting little additive genetic variation in acid stress tolerance. The results suggest that although local genetic adaptation to acid stress has taken place, the current variation in acid stress tolerance in acidified populations may owe largely to non-genetic effects. However, low but significant heritabilities (h ² 0.07–0.22) in all traits – including viability itself – under a wide range of pH conditions suggests that environmental stress created by low pH is unlikely to lower moor frog populations' ability to respond to selection in the traits studied. Nevertheless, acid conditions could lower populations' ability to respond to selection in the long run through reduction in effective population size.     

Functional aspects of root architecture and mycorrhizal inoculation with respect to nutrient uptake capacity

The aim of this research was to investigate the effect of arbuscular mycorrhizal (AM) colonisation on root morphology and nitrogen uptake capacity of carob ( Ceratonia siliqua L.) under high and low nutrient conditions. The experimental design was a factorial arrangement of presence/absence of mycorrhizal fungus inoculation ( Glomus intraradices) and high/low nutrient status. Percent AM colonisation, nitrate and ammonium uptake capacity, and nitrogen and phosphorus contents were determined in 3-month-old seedlings. Grayscale and colour images were used to study root morphology and topology, and to assess the relation between root pigmentation and physiological activities. AM colonisation lead to a higher allocation of biomass to white and yellow parts of the root. Inorganic nitrogen uptake capacity per unit root length and nitrogen content were greatest in AM colonised plants grown under low nutrient conditions. A better match was found between plant nitrogen content and biomass accumulation, than between plant phosphorus content and biomass accumulation. It is suggested that the increase in nutrient uptake capacity of AM colonised roots is dependent both on changes in root morphology and physiological uptake potential. This study contributes to an understanding of the role of AM fungi and root morphology in plant nutrient uptake and shows that AM colonisation improves the nitrogen nutrition of plants, mainly when growing at low levels of nutrients.     

Mycorrhizal Symbiosis and Local Adaptation in Aster amellus: A Field Transplant Experiment

Many plant populations have adapted to local soil conditions. However, the role of arbuscular mycorrhizal fungi is often overlooked in this context. Only a few studies have used reciprocal transplant experiments to study the relationships between soil conditions, mycorrhizal colonisation and plant growth. Furthermore, most of the studies were conducted under controlled greenhouse conditions. However, long-term field experiments can provide more realistic insights into this issue. We conducted a five-year field reciprocal transplant experiment to study the relationships between soil conditions, arbuscular mycorrhizal fungi and plant growth in the obligate mycotrophic herb Aster amellus. We conducted this study in two regions in the Czech Republic that differ significantly in their soil nutrient content, namely Czech Karst (region K) and Ceske Stredohori (region S). Plants that originated from region S had significantly higher mycorrhizal colonisation than plants from region K, indicating that the percentage of mycorrhizal colonisation has a genetic basis. We found no evidence of local adaptation in Aster amellus. Instead, plants from region S outperformed the plants from region K in both target regions. Similarly, plants from region S showed more mycorrhizal colonisation in all cases, which was likely driven by the lower nutrient content in the soil from that region. Thus, plant aboveground biomass and mycorrhizal colonisation exhibited corresponding differences between the two target regions and regions of origin. Higher mycorrhizal colonisation in the plants from region with lower soil nutrient content (region S) in both target regions indicates that mycorrhizal colonisation is an adaptive trait. However, lower aboveground biomass in the plants with lower mycorrhizal colonisation suggests that the plants from region K are in fact maladapted by their low inherent mycorrhizal colonization. We conclude that including mycorrhizal symbiosis in local adaptation studies may increase our understanding of the mechanisms by which plants adapt to their environment.     

The highly dynamic CRISPR1 system of Streptococcus agalactiae controls the diversity of its mobilome

Clustered regularly interspaced short palindromic repeats (CRISPR) confer immunity against mobile genetic elements (MGEs) in prokaryotes. Streptococcus agalactiae, a leading cause of neonatal infections contains in its genome two CRISPR/Cas systems. We show that type 1‐C CRISPR2 is present in few strains but type 2‐A CRISPR1 is ubiquitous. Comparative sequence analysis of the CRISPR1 spacer content of 351 S. agalactiae strains revealed that it is extremely diverse due to the acquisition of new spacers, spacer duplications and spacer deletions that witness the dynamics of this system. The spacer content profile mirrors the S. agalactiae population structure. Transfer of a conjugative transposon targeted by CRISPR1 selected for spacer rearrangements, suggesting that deletions and duplications pre‐exist in the population. The comparison of protospacers located within MGE or the core genome and protospacer‐associated motif‐shuffling demonstrated that the GG motif is sufficient to discriminate self and non‐self and for spacer selection and integration. Strikingly more than 40% of the 949 different CRISPR1 spacers identified target MGEs found in S. agalactiae genomes. We thus propose that the S. agalactiae type II‐A CRISPR1/Cas system modulates the cohabitation of the species with its mobilome, as such contributing to the diversity of MGEs in the population.     

A proteomic investigation of Streptococcus agalactiae reveals that human serum induces the C protein β antigen and arginine deiminase

Streptococcus agalactiae is a major neonatal pathogen. Disease progression is characterised by bacterial adaptation from commensal maternal vaginal colonisation to environments associated with neonatal disease, including exposure to blood. To explore this adaptation in vitro, we have used proteomics to identify proteins differentially expressed following growth on Todd Hewitt agar in the presence or absence of 10% v/v human serum. Twelve differentially expressed proteins were identified. Notably, the C protein β antigen and arginine deiminase proteins were upregulated following growth in the presence of human serum, consistent with previous studies implicating these two proteins in the pathogenesis of S. agalactiae disease.     

A gastrointestinal nematode in pregnant and lactating mice alters maternal and neonatal microbiomes

The maternal microbiome is understood to be the principal source of the neonatal microbiome but the consequences of intestinal nematodes on pregnant and lactating mothers and implications for the neonatal microbiome are unknown. Using pregnant CD1 mice infected with Heligmosomoides bakeri, we investigated the microbiomes in maternal tissues (intestine, vagina, and milk) and in the neonatal stomach using MiSeq sequencing of bacterial 16S rRNA genes. Our first hypothesis was that maternal nematode infection altered the maternal intestinal, vaginal, and milk microbiomes and associated metabolic pathways. Maternal nematode infection was associated with increased beta-diversity and abundance of fermenting bacteria as well as Lactobacillus in the maternal caecum 2 days after parturition, together with down-regulated carbohydrate, amino acid and vitamin biosynthesis pathways. Maternal nematode infection did not alter the vaginal or milk microbiomes. Our second hypothesis was that maternal infection would shape colonization of the neonatal microbiome. Although the pup stomach microbiome was similar to that of the maternal vaginal microbiome, pups of infected dams had higher beta-diversity at day 2, and a dramatic expansion in the abundance of Lactobacillus between days 2 and 7 compared with pups nursing uninfected dams. Our third hypothesis that maternal nematode infection altered the composition of neonatal microbiomes was confirmed as we observed up-regulation of several putatively beneficial microbial pathways associated with synthesis of essential and branched-chain amino acids, vitamins, and short-chain fatty acids. We believe this is the first study to show that a nematode living in the maternal intestine is associated with altered composition and function of the neonatal microbiome.     

Biological characteristics of an invasive south African species

Senecio inaequidens, a south African species that has recently invaded parts of Europe, especially human disturbed habitats, was examined under laboratory and glasshouse conditions to assess the germination of achenes, soil seed banks, growth of seedlings and its breeding system. These observations allowed identification of ecological characteristics that may contribute to the invasiveness of the species. Achenes derived from three populations of S. inaequidens were germinated over a wide temperature range (from 14/6 °C day/night temperatures to a constant temperature of 30 °C). They exhibited a rapid achene germination especially when temperatures reached 20 °C. Germination of seeds from soil samples also exhibited a prolonged germination over time suggesting therefore different types of dormancy. Growth response was studied along a nitrogen gradient from 0.33 to 16.0 mmol l^–1 and data were interpreted using growth analysis. Seedlings of S. inaequidens exhibited a slow growth in low nutrient availability and a rapid growth rate when exposed to high nutrient availability, which contributed to producing a large leaf area and biomass. Four types of pollination were studied in S. inaequidens. Hand-pollinations showed that S. inaequidens was mostly self-incompatible. Self-pollinated capitula only showed 7.6% viable achenes, significantly lower than insect-pollinated and cross-pollinated capitula. Some individuals were more self-fertile than others ensuring the production of some offspring in a situation of colonisation. Natural pollination occurred with generalist insects. Most of these results confirm the invasive ability of S. inaequidens.     

Organic enrichment of sediments reduces arbuscular mycorrhizal fungi in oligotrophic lake plants

1. Arbuscular mycorrhizal fungi (AMF) commonly colonise isoetid species inhabiting oxygenated sediments in oligotrophic lakes but are usually absent in other submerged plants. We hypothesised that organic enrichment of oligotrophic lake sediments reduces AMF colonisation and hyphal growth because of sediment O₂ depletion and low carbon supply from stressed host plants. 2. We added organic matter to sediments inhabited by isoetids and measured pore‐water chemistry (dissolved O₂, inorganic carbon, Fe²⁺ and ), colonisation intensity of roots and hyphal density after 135 days of exposure. 3. Addition of organic matter reduced AMF colonisation of roots of both Lobelia dortmanna and Littorella uniflora, and high additions stressed the plants. Even small additions of organic matter almost stopped AMF colonisation of initially un‐colonised L. uniflora, though without reducing plant growth. Mean hyphal density in sediments was high (6 and 15 m cm^?3) and comparable with that in terrestrial soils (2–40 m cm^?3). Hyphal density was low in the upper 1 cm of isoetid sediments, high in the main root zone between 1 and 8 cm and positively related to root density. Hyphal surface area exceeded root surface area by 1.7–3.2 times. 4. We conclude that AMF efficiently colonise isoetids in oligotrophic sediments and form extensive hyphal networks. Small additions of organic matter to sediments induce sediment anoxia and reduce AMF colonisation of roots but cause no apparent plant stress. High organic addition induces night‐time anoxia in both the sediment and the plant tissue. Tissue anoxia reduces root growth and AMF colonisation, probably because of restricted translocation of nutrient ions and organic solutes between roots and leaves. Isoetids should rely on AMF for P uptake on nutrient‐poor mineral sediments but are capable of growing without AMF on organic sediments.     

Positive effects of proline addition on the central metabolism of wild-type and lactic acid-producing <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains

In Saccharomyces cerevisiae, proline is a stress protectant interacting with other substrate uptake systems against oxidative stress under low pH conditions. In this study, we performed metabolomics analysis to investigate the response associated with an increase in cell growth rates and maximum densities when cells were treated with proline under normal and acid stress conditions. Metabolome data show that concentrations of components of central metabolism are increased in proline-treated S. cerevisiae. No consumption of proline was observed, suggesting that proline does not act as a nutrient but regulates metabolic state and growth of cells. Treatment of lactic acid-producing yeast with proline during lactic acid bio-production improved growth rate and increased the final concentration of lactic acid.     

Interactions between ammonium and pH of the nutrient solution supplied to gerbera (Gerbera jamesonii) grown in pumice

The interactions between the NH₄-N/total-N supply ratio (N _r) and the pH of the nutrient solution delivered to the plants regarding the nutrient availability and its influence on plant growth and yield were investigated in a gerbera crop grown in pumice. Four treatments obtained by combining a low and a high N _r (0.02 and 0.13 on a molar basis, respectively) with two different levels of nutrient solution pH (5.0 and 5.8, respectively) were compared in a long-term glasshouse experiment. The low N _r resulted in significantly higher pH levels in the rhizosphere, as indicated by the values measured in the drainage water. A high nutrient solution pH further increased pH in the root zone at low N _r but had no influence on it at the higher N _r level. The high pH in the root zone restricted significantly the Cu, Mn, and Zn uptake as indicated by both analytical results and visual symptoms, which were typical for Cu- and Mn-deficiencies. Mn-deficiency was induced only when a low N _r was combined with a high nutrient solution pH. The Cu-deficiency symptoms appeared earlier, were more severe and occurred also when the low N _r was combined with a low nutrient solution pH, but to a lesser extent. As a result of these nutrient deficiencies, both the growth and the flower yield were severely restricted when N _r was low. These results indicate that gerbera is prone to Cu- and Mn deficiencies at pH values above 6 in the rhizosphere. The maintenance of root zone pH below this level in soilless culture is most successfully attained by properly adjusting the NH₄-N/total-N supply ratio rather than the pH of the nutrient solution delivered to the plants.     

Alkalinity Tolerance of Woody Species Used in Bauxite Waste Rehabilitation, Western Australia

Glasshouse trials, using trickle irrigation and increasing levels of NaOH-induced alkalinity, identified species that could be expected to tolerate the high-pH conditions of bauxite processing waste residue sites. Of 29 taxa tested, the most tolerant were Casuarina obesa, Melaleuca lanceolata, M. armillaris, M. nesophila, Eucalyptus loxophleba, E. halophila, E. platypus, Tamarix aphylla, and a particular clone of E. camaldulensis; E. spathulata, E. tetragona, E. preissiana, E. gomphocephala, E. diptera, and E. occidentalis proved to be relatively sensitive to severe alkaline conditions. Tolerance appeared to relate to an ability to maintain root membrane function, nutrient uptake balance, and ultimately root tissue structure while under increasing levels of alkalinity stress. Species normally inhabiting alkaline soils tended to have increased growth rates in nutrient irrigation conditions between pH 8 and 10 compared with control plants irrigated with nutrient solutions of pH values near 7.4. However, once the irrigation solutions reached pH 12 and the buffering capacity of the soil appeared to be exceeded, the condition of susceptible plants rapidly declined and death followed. Sensitive plants initially showed symptoms related to nutrient deficiency, followed by wilting and death as the root systems failed. Field trial conditions in the bauxite residue impoundments at Kwinana, Western Australia, include soils with pH values as high as 11.00. In general, the relative survival and growth of seedlings after eight months were predicted by the response under glasshouse trial conditions. Appropriately designed stress trials can be important ecological techniques in choosing species most capable of surviving difficult environmental conditions in the rehabilitation of damaged landscapes.     

Analysis of expression and regulatory functions of the ribosome-binding protein TypA in Mycobacterium tuberculosis under stress conditions

In many bacterial species, the translational GTPase TypA acts as a global stress- and virulence regulator and also mediates resistance to the antimicrobial peptide BPI. On the chromosome of M. tuberculosis, typA is located next to narGHJI, which plays a role in adaptation of the pathogen to various environmental conditions. Here, we show that Mycobacterium tuberculosis is sensitive to P2, a derivative of BPI. Using a typA mutant of M. tuberculosis, we found this phenotype to be independent of TypA. We further tested typA expression in M. tuberculosis under defined stress conditions, such as oxygen- and nutrient depletion, low pH, heat shock, antibiotic stress and the presence of P2, and found that typA expression remains unaffected by any of these conditions. Analysis of growth and whole-genome expression revealed similar growth kinetics and gene expression profiles of the wild type and the mutant under normal growth conditions as well as under stress conditions. Our results suggest that in contrast to the findings in other bacteria, TypA does not act as a global stress- and virulence regulator in M. tuberculosis.     

Acid tolerance inLeuconostoc oenos. Isolation and characterization of an acid-resistant mutant

The acid tolerance ofLeuconostoc oenos was examined in cells surviving at pH 2.6, which is lower than the acid limit of growth (about pH 3.0). Acid-adapted cells survived better than non-adapted cells. Tolerance to acid stress was found to be dependent upon the adaptive pH. Acid resistance was increased by an order of magnitude for cultures adapted to a pH of about 2.9. Inhibiting protein synthesis with chloramphenicol prior to acid shock revealed that acid adaptation may involve two separate systems, one of which appears to be independent of protein synthesis. The acid-resistant mutant LoV8413, isolated during a long-term survival screen at pH 2.6, was found to be able to grow in acidic media and was characterized by a high H⁺-ATPase activity at low pH. The data from electrophoretic analysis of total proteins labeled with [³⁵S] methionine indicate that large amounts of a protein of 42 kDa molecular mass were produced within this acid-resistant mutant.