Bacillus subtilis forms twisted cells with cell wall integrity defects upon removal of the molecular chaperones DnaK and trigger factor

The protein homeostasis network ensures a proper balance between synthesis, folding, and degradation of all cellular proteins. DnaK and trigger factor (TF) are ubiquitous bacterial molecular chaperones that assist in protein folding, as well as preventing protein misfolding and aggregation. In Escherichia coli, DnaK and TF possess partially overlapping functions. Their combined depletion results in proteostasis collapse and is synthetically lethal at temperatures above 30°C. To increase our understanding on how proteostasis is maintained in Gram-positive bacteria, we have investigated the physiological effects of deleting dnaK and tig (encoding for DnaK and TF) in Bacillus subtilis. We show that combined deletion of dnaK and tig in B. subtilis is non-lethal, but causes a severe pleiotropic phenotype, including an aberrant twisted and filamentous cell morphology, as well as decreased tolerance to heat and to cell wall active antibiotics and hydrolytic enzymes, indicative of defects in cell wall integrity. In addition, cells lacking DnaK and TF have a much smaller colony size due to defects in motility. Despite these physiological changes, we observed no major compromises in important cellular processes such as cell growth, FtsZ localization and division and only moderate defects in spore formation. Finally, through suppressor analyses, we found that the wild-type cell shape can be partially restored by mutations in genes involved in metabolism or in other diverse cellular processes

Bistable differentiation in an isogenic cell population

PhD ThesisSingle-cell organisms such as bacteria have traditionally been regarded as discrete units, which in turn has been reflected by the bulk-level methods used to study them. A growing culture of the bacterium Bacillus subtilis will exhibit a range of heterogeneous genetic developmental programmes such as motility, competence, and finally sporulation. As a popular choice for production of compounds in bioreactors, the bistable behaviours of B. subtilis may be undesirable traits, as they divert resources from their intended activity of synthesising a product. This thesis investigates a novel observation that expression of a ribosomal subunit gene (rpsD) is elevated in the non-motile state of B. subtilis, using unstable GFP reporter constructs. The implications of using a proteolytically unstable protein as a reporter are also investigated with regard to the effect of protein degradation rates on the reporter construct, as well as presenting evidence for modulation of ClpXP activity in a pnpA background. Investigation of the motile/non-motile heterogeneous phenotype of B. subtilis posed a challenge for automated analysis pipelines. This thesis addresses this problem by developing and testing microscopy analysis pipelines designed to circumvent the traditional requirement for physically separated objects in a phase contrast channel, and instead using nucleoid or membrane stains to identify cells in a microscopy image. Other factors impacting the activity of a proteolytically unstable PrpsD reporter construct were investigated, including the rate of degradation of the reporter, and integration locus of the reporter construct. To assess the impact of locus positioning, a genetic tool was also created to survey changes in noise and overall expression levels from two homogeneously expressed promoters across different positions on the chromosome.Biotechnology and Biological Sciences Research Council (BBSRC

Isolation and Identification of Bioactive Secondary Metabolites

The health benefits of food, plants, fruits, and seaweeds stem from the biological activities of their constituents—namely, secondary metabolites. The study of secondary metabolites and their potential to treat and/or prevent a number of diseases has become a research topic of growing interest for biologists, pharmacists, and chemists. Notably, in order to propose a compound as a potential new drug with pharmacological effects, the chemical structure of this compound and its biological activity against a given target must be well established. The Special Issue, “Isolation and Identification of Bioactive Secondary Metabolites”, considers species beyond their nutritional value and identifies instances of wider and more efficient use, thereby contributing to a more sustainable management of natural resources. The fifteen articles published in this Special Issue reflect the latest research trends, and consider the isolation, identification, and assessment of the beneficial effects of secondary metabolites from both edible and inedible species. Thus, these contributions collectively demonstrate that these compounds, and their plants of origin, should be valued beyond their nutritional benefits

Co-existence and niche separation of two subspecies of Photorhabdus temperata associated with Heterorhabditis downesi in a dune grassland

Entomopathogenic nematodes belonging to the families Heterorhabditidae and Steinernematidae form symbiotic relationships with entomopathogenic bacteria from the genera Photorhabdus and Xenorhabdus, respectively. Previous studies showed that Heterorhabditis downesi was associated with two different colour variants (pur and yel) of its Photorhabdus bacterial symbiont in sand dunes on North Bull Island in Dublin Bay, with evidence of the pur variant being more common to the front of the dunes. The main objectives of this study were to confirm the distribution of both colour variants of the nematode-bacterium complex on North Bull Island, identify them, investigate the specificity of the nematode-bacterium relationship and ascertain characteristics of the bacterial variants that might explain their distribution. A section of the dune system on North Bull Island was surveyed in 2008 and resurveyed in 2012 (Chapter 2). In 2008 the colour variants were spatially segregated, with nematodes associated with the pur variant recovered from the front section of the dunes only and nematodes associated with the yel variant recovered from the rear section only. In 2012 nematodes associated with each colour variant were recovered from the front section of the dunes and only nematodes associated with the yel variant were recovered from the rear section of the dunes. The nematode-pur complex was more abundant than the nematode-yel complex in sections where both combinations co-occurred. Three laboratory lines of each nematode and its associated bacterial colour variant were established for further study. A phylogenetic analysis of the nematode ITS region showed that all the nematode isolates were monophyletic, with no difference between the isolates carrying the pur or yel bacterial symbionts. On the other hand, a phylogenetic analysis of a portion of the gyrB gene of the bacterial isolates showed that the two colour variants were distinct subspecies, Photorhabdus temperata subsp. temperata (yel) and Photorhabdus temperata subsp. cinerea (pur). This confirms Photorhabdus temperata subsp. temperata as the bacterial partner in the YEL H. downesi – Photorhabdus complex. In addition, this is the first report of Photorhabdus temperata subsp. cinerea in Ireland and also one of the few reports of the co-occurrence of two different symbiotic bacteria subspecies with a single nematode species (Chapter 2). In antibiosis tests against a range of organisms the P. temperata subsp. cinerea isolates had a greater inhibitory effect than the P. temperata subsp. temperata isolates. Only one pair of isolates, yel1 and pur2, showed any antibiotic activity against the other subspecies and this pair were mutually inhibitory (Chapter 2). Phenotypic tests indicated that the subspecies had different growth requirements as the pur isolates grew better on MacConkey agar than on nutrient agar while there was no difference between the two media for the yel isolates. Biochemical characterisation was carried out using API 20E test strips. There was variability both within and between the colour variants, however a notable difference was that only the yel isolates were able to utilise trehalose, a major component of insect hemolymph (Chapter 2). The cross-compatibility of the two different bacterial subspecies with the heterogeneous nematode isolates was tested by injecting Galleria mellonella larvae with 10,000 cells of either the pur1 or yel3 isolate or both and then infecting them with H. downesi nematodes carrying either pur1 or yel3 bacteria. The emerging nematodes carried the bacterial subspecies that had been injected into the host, irrespective of which bacterial subspecies the infecting nematode had carried and hybrid lines of nematode-bacterium complex were successfully maintained for the duration of this study (Chapter 3). In the case of cadavers infected with both bacterial types all the emerging nematodes tended to carry a single subspecies. However, between 20 % and 30 % of the cadavers produced some nematodes carrying the pur1 and some carrying the yel3 isolate, (indicating that both bacterial subspecies were colonising the insect host and subsequently colonising the developing nematodes prior to emergence) and where this occurred a higher proportion of the nematodes carried the yel3 isolate. Furthermore, there was an apparent shift in the colonisation of the nematodes within the host cadaver the proportion of nematodes carrying the yel3 isolate was higher in later emerging nematodes (Chapter 3). To test the hypothesis that the distribution of the colour variants was due to host specialisation (H. downesi complex differs from front to back of the dune system) the pathogenicity of the homogenous and heterogenous combinations against Hylobius abietis larvae was investigated. The LD50 was significantly higher for the homogenous YEL/yel combination compared to the heterogeneous YEL/pur combination. There was no difference in reproduction rate of the nematodes carrying either subspecies in either H. abietis or G. mellonella and this was backed up by tests of all three isolates of each colour type. All six isolates from North Bull Island were tested against insects from three orders (Lepidoptera, Coleoptera (2) and Diptera), with no differences in mortality or reproduction rate. This is the first report of EPN being able to use the novel host kelp fly (Coelopa spp.). While there was no difference in mortality rates between the PUR and YEL strains in this host there did appear to be a difference in the rate of progression of the infection with nematodes carrying the yel bacteria emerging earlier than those carrying the pur bacteria (Chapter 4). To test the hypothesis that the bacteria influenced nematode survival in desiccated hosts G. mellonella larvae were infected with the homogenous and heterogeneous combinations and stored in dry sand or at 0% relative humidity. There was no difference due to bacteria type in the number of cadavers with emergence from those stored in dry sand for 28 days. There was a highly significant difference due to bacteria type in the number of cadavers with emergence for cadavers stored at 0 % relative humidity for 28 days, with emergence from more cadaver infected with the pur1 isolate. Overall, more nematodes emerged from cadavers infected with the pur1 isolate following 28 days storage in dry sand or at 0 % relative humidity (Chapter 5). Heterorhabditis downesi is associated with two different subspecies of Photorhabdus temperata on North Bull Island: Photorhabdus temperata subsp. temperata and Photorhabdus temperata subsp. cinerea and the association of the nematode with a particular subspecies appears to vary with the soil conditions prevalent in the front and the rear sections of the dunes: Under normal conditions P.t. temperata appears to outcompete P.t. cinerea in forming a symbiotic association with the nematode when both subspecies are present in the same insect host, resulting in the H. downesi-P.t. temperata association predominating in the rear section of the dunes, where the increased organic content of the soil favours moisture retention. On the other hand the drier conditions that predominate in the sandier soil towards the front of the dune system favour H. downesi-P.t. cinerea association

Co-existence and niche separation of two subspecies of Photorhabdus temperata associated with Heterorhabditis downesi in a dune grassland

Entomopathogenic nematodes belonging to the families Heterorhabditidae and Steinernematidae form symbiotic relationships with entomopathogenic bacteria from the genera Photorhabdus and Xenorhabdus, respectively. Previous studies showed that Heterorhabditis downesi was associated with two different colour variants (pur and yel) of its Photorhabdus bacterial symbiont in sand dunes on North Bull Island in Dublin Bay, with evidence of the pur variant being more common to the front of the dunes. The main objectives of this study were to confirm the distribution of both colour variants of the nematode-bacterium complex on North Bull Island, identify them, investigate the specificity of the nematode-bacterium relationship and ascertain characteristics of the bacterial variants that might explain their distribution. A section of the dune system on North Bull Island was surveyed in 2008 and resurveyed in 2012 (Chapter 2). In 2008 the colour variants were spatially segregated, with nematodes associated with the pur variant recovered from the front section of the dunes only and nematodes associated with the yel variant recovered from the rear section only. In 2012 nematodes associated with each colour variant were recovered from the front section of the dunes and only nematodes associated with the yel variant were recovered from the rear section of the dunes. The nematode-pur complex was more abundant than the nematode-yel complex in sections where both combinations co-occurred. Three laboratory lines of each nematode and its associated bacterial colour variant were established for further study. A phylogenetic analysis of the nematode ITS region showed that all the nematode isolates were monophyletic, with no difference between the isolates carrying the pur or yel bacterial symbionts. On the other hand, a phylogenetic analysis of a portion of the gyrB gene of the bacterial isolates showed that the two colour variants were distinct subspecies, Photorhabdus temperata subsp. temperata (yel) and Photorhabdus temperata subsp. cinerea (pur). This confirms Photorhabdus temperata subsp. temperata as the bacterial partner in the YEL H. downesi – Photorhabdus complex. In addition, this is the first report of Photorhabdus temperata subsp. cinerea in Ireland and also one of the few reports of the co-occurrence of two different symbiotic bacteria subspecies with a single nematode species (Chapter 2). In antibiosis tests against a range of organisms the P. temperata subsp. cinerea isolates had a greater inhibitory effect than the P. temperata subsp. temperata isolates. Only one pair of isolates, yel1 and pur2, showed any antibiotic activity against the other subspecies and this pair were mutually inhibitory (Chapter 2). Phenotypic tests indicated that the subspecies had different growth requirements as the pur isolates grew better on MacConkey agar than on nutrient agar while there was no difference between the two media for the yel isolates. Biochemical characterisation was carried out using API 20E test strips. There was variability both within and between the colour variants, however a notable difference was that only the yel isolates were able to utilise trehalose, a major component of insect hemolymph (Chapter 2). The cross-compatibility of the two different bacterial subspecies with the heterogeneous nematode isolates was tested by injecting Galleria mellonella larvae with 10,000 cells of either the pur1 or yel3 isolate or both and then infecting them with H. downesi nematodes carrying either pur1 or yel3 bacteria. The emerging nematodes carried the bacterial subspecies that had been injected into the host, irrespective of which bacterial subspecies the infecting nematode had carried and hybrid lines of nematode-bacterium complex were successfully maintained for the duration of this study (Chapter 3). In the case of cadavers infected with both bacterial types all the emerging nematodes tended to carry a single subspecies. However, between 20 % and 30 % of the cadavers produced some nematodes carrying the pur1 and some carrying the yel3 isolate, (indicating that both bacterial subspecies were colonising the insect host and subsequently colonising the developing nematodes prior to emergence) and where this occurred a higher proportion of the nematodes carried the yel3 isolate. Furthermore, there was an apparent shift in the colonisation of the nematodes within the host cadaver the proportion of nematodes carrying the yel3 isolate was higher in later emerging nematodes (Chapter 3). To test the hypothesis that the distribution of the colour variants was due to host specialisation (H. downesi complex differs from front to back of the dune system) the pathogenicity of the homogenous and heterogenous combinations against Hylobius abietis larvae was investigated. The LD50 was significantly higher for the homogenous YEL/yel combination compared to the heterogeneous YEL/pur combination. There was no difference in reproduction rate of the nematodes carrying either subspecies in either H. abietis or G. mellonella and this was backed up by tests of all three isolates of each colour type. All six isolates from North Bull Island were tested against insects from three orders (Lepidoptera, Coleoptera (2) and Diptera), with no differences in mortality or reproduction rate. This is the first report of EPN being able to use the novel host kelp fly (Coelopa spp.). While there was no difference in mortality rates between the PUR and YEL strains in this host there did appear to be a difference in the rate of progression of the infection with nematodes carrying the yel bacteria emerging earlier than those carrying the pur bacteria (Chapter 4). To test the hypothesis that the bacteria influenced nematode survival in desiccated hosts G. mellonella larvae were infected with the homogenous and heterogeneous combinations and stored in dry sand or at 0% relative humidity. There was no difference due to bacteria type in the number of cadavers with emergence from those stored in dry sand for 28 days. There was a highly significant difference due to bacteria type in the number of cadavers with emergence for cadavers stored at 0 % relative humidity for 28 days, with emergence from more cadaver infected with the pur1 isolate. Overall, more nematodes emerged from cadavers infected with the pur1 isolate following 28 days storage in dry sand or at 0 % relative humidity (Chapter 5). Heterorhabditis downesi is associated with two different subspecies of Photorhabdus temperata on North Bull Island: Photorhabdus temperata subsp. temperata and Photorhabdus temperata subsp. cinerea and the association of the nematode with a particular subspecies appears to vary with the soil conditions prevalent in the front and the rear sections of the dunes: Under normal conditions P.t. temperata appears to outcompete P.t. cinerea in forming a symbiotic association with the nematode when both subspecies are present in the same insect host, resulting in the H. downesi-P.t. temperata association predominating in the rear section of the dunes, where the increased organic content of the soil favours moisture retention. On the other hand the drier conditions that predominate in the sandier soil towards the front of the dune system favour H. downesi-P.t. cinerea association