Computational Strategies for the Identification of a Transcriptional Biomarker Panel to Sense Cellular Growth States in Bacillus subtilis.

A goal of the biotechnology industry is to be able to recognise detrimental cellular states that may lead to suboptimal or anomalous growth in a bacterial population. Our current knowledge of how different environmental treatments modulate gene regulation and bring about physiology adaptations is limited, and hence it is difficult to determine the mechanisms that lead to their effects. Patterns of gene expression, revealed using technologies such as microarrays or RNA-seq, can provide useful biomarkers of different gene regulatory states indicative of a bacterium's physiological status. It is desirable to have only a few key genes as the biomarkers to reduce the costs of determining the transcriptional state by opening the way for methods such as quantitative RT-PCR and amplicon panels. In this paper, we used unsupervised machine learning to construct a transcriptional landscape model from condition-dependent transcriptome data, from which we have identified 10 clusters of samples with differentiated gene expression profiles and linked to different cellular growth states. Using an iterative feature elimination strategy, we identified a minimal panel of 10 biomarker genes that achieved 100% cross-validation accuracy in predicting the cluster assignment. Moreover, we designed and evaluated a variety of data processing strategies to ensure our methods were able to generate meaningful transcriptional landscape models, capturing relevant biological processes. Overall, the computational strategies introduced in this study facilitate the identification of a detailed set of relevant cellular growth states, and how to sense them using a reduced biomarker panel.

Impact of Redox Conditions on Antibiotic Resistance Conjugative Gene Transfer Frequency and Plasmid Fate in Wastewater Ecosystems.

Wastewater is a common pathway for the spread of antibiotic resistance (AR) genes and bacteria into the environment. Biological treatment can mitigate this path, but horizontal gene transfer (HGT) between bacteria also occurs in such processes, although the influence of bioreactor habitat and ecology on HGT frequency is not well understood. Here, we quantified how oxidation-reduction (redox) conditions impact the fate of a Green fluorescent protein (Gfp)-tagged AR plasmid (pRP4-gfp) within an E. coli host (EcoFJ1) in the liquid phase and biofilms in bioreactors. Replicate reactors treating domestic wastewater were operated under stable aerobic (+195 ± 25 mV), anoxic (-15 ± 50 mV), and anaerobic (-195 ± 15 mV) conditions, and flow cytometry and selective plating were used to quantify donor strain, EcoFJ1(pRP4-gfp), and putative transconjugants over time. Plasmid pRP4-gfp-bearing cells disappeared rapidly in aerobic ecosystems (∼2.0 log reduction after 72 h), especially in the liquid phase. In contrast, EcoFJ1(pRP4-gfp) and putative transconjugants persisted much longer in anaerobic biofilms (∼1.0 log reduction, after 72 h). Plasmid transfer frequencies were also higher under anaerobic conditions. In parallel, protozoan abundances were over 20 times higher in aerobic reactors relative to anaerobic reactors, and protozoa numbers significantly inversely correlated with pRP4-gfp signals across all reactors (p < 0.05). Taken together, observed HGT frequency and plasmid retention are impacted by habitat conditions and trophic effects, especially oxygen conditions and apparent predation. New aerobic bioreactor designs are needed, ideally employing passive aeration to save energy, to minimize resistance HGT in biological wastewater treatment processes.

Proteomic analysis of Bacillus subtilis strains engineered for improved production of heterologous proteins.

The use of bacterial systems for recombinant protein production has advantages of simplicity, time and cost over competing systems. However, widely used bacterial expression systems (e.g. Escherichia coli, Pseudomonas fluorescens) are not able to secrete soluble proteins directly into the culture medium. This limits yields and increases downstream processing time and costs. In contrast, Bacillus spp. secrete native enzymes directly into the culture medium at grams-per-litre quantities, although the yields of some recombinant proteins are severely limited. We have engineered the Bacillus subtilis genome to generate novel strains with precise deletions in the genes encoding ten extracytoplasmic proteases that affect recombinant protein secretion, which lack chromosomal antibiotic resistance genes. The deletion sites and presence of single nucleotide polymorphisms were confirmed by sequencing. The strains are stable and were used in industrial-scale fermenters for the production of the Bacillus anthracis vaccine protein, protective antigen, the productivity of which is extremely low in the unmodified strain. We also show that the deletion of so-called quality control proteases appears to influence cell-wall synthesis, resulting in the induction of the cell-wall stress regulon that encodes another quality control protease.

Extracytoplasmic proteases determining the cleavage and release of secreted proteins, lipoproteins, and membrane proteins in Bacillus subtilis.

Gram-positive bacteria are known to export many proteins to the cell wall and growth medium, and accordingly, many studies have addressed the respective protein export mechanisms. In contrast, very little is known about the subsequent fate of these proteins. The present studies were therefore aimed at determining the fate of native exported proteins in the model organism Bacillus subtilis. Specifically, we employed a gel electrophoresis-based liquid chromatography-mass spectrometry approach to distinguish the roles of the membrane-associated quality control proteases HtrA and HtrB from those of eight other proteases that are present in the cell wall and/or growth medium of B. subtilis. Notably, HtrA and HtrB were previously shown to counteract potentially detrimental "protein export stresses" upon overproduction of membrane or secreted proteins. Our results show that many secreted proteins, lipoproteins, and membrane proteins of B. subtilis are potential substrates of extracytoplasmic proteases. Moreover, potentially important roles of HtrA and HtrB in the folding of native secreted proteins into a protease-resistant conformation, the liberation of lipoproteins from the membrane-cell wall interface, and the degradation of membrane proteins are uncovered. Altogether, our observations show that HtrA and HtrB are crucial for maintaining the integrity of the B. subtilis cell even under nonstress conditions.

A model industrial workhorse: Bacillus subtilis strain 168 and its genome after a quarter of a century.

The vast majority of genomic sequences are automatically annotated using various software programs. The accuracy of these annotations depends heavily on the very few manual annotation efforts that combine verified experimental data with genomic sequences from model organisms. Here, we summarize the updated functional annotation of Bacillus subtilis strain 168, a quarter century after its genome sequence was first made public. Since the last such effort 5 years ago, 1168 genetic functions have been updated, allowing the construction of a new metabolic model of this organism of environmental and industrial interest. The emphasis in this review is on new metabolic insights, the role of metals in metabolism and macromolecule biosynthesis, functions involved in biofilm formation, features controlling cell growth, and finally, protein agents that allow class discrimination, thus allowing maintenance management, and accuracy of all cell processes. New 'genomic objects' and an extensive updated literature review have been included for the sequence, now available at the International Nucleotide Sequence Database Collaboration (INSDC: AccNum AL009126.4).

The iron-binding protein Dps2 confers peroxide stress resistance on Bacillus anthracis.

Iron is an essential nutrient that is implicated in most cellular oxidation reactions. However, iron is a highly reactive element that, if not appropriately chaperoned, can react with endogenously and exogenously generated oxidants such as hydrogen peroxide to generate highly toxic hydroxyl radicals. Dps proteins (DNA-binding proteins from starved cells) form a distinct class (the miniferritins) of iron-binding proteins within the ferritin superfamily. Bacillus anthracis encodes two Dps-like proteins, Dps1 and Dps2, the latter being one of the main iron-containing proteins in the cytoplasm. In this study, the function of Dps2 was characterized in vivo. A B. anthracis Δdps2 mutant was constructed by double-crossover mutagenesis. The growth of the Δdps2 mutant was unaffected by excess iron or iron-limiting conditions, indicating that the primary role of Dps2 is not that of iron sequestration and storage. However, the Δdps2 mutant was highly sensitive to H(2)O(2), and pretreatment of the cells with the iron chelator deferoxamine mesylate (DFM) significantly reduced its sensitivity to H(2)O(2) stress. In addition, the transcription of dps2 was upregulated by H(2)O(2) treatment and derepressed in a perR mutant, indicating that dps2 is a member of the regulon controlled by the PerR regulator. This indicates that the main role of Dps2 is to protect cells from peroxide stress by inhibiting the iron-catalyzed production of OH.

Cellular iron distribution in Bacillus anthracis.

Although successful iron acquisition by pathogens within a host is a prerequisite for the establishment of infection, surprisingly little is known about the intracellular distribution of iron within bacterial pathogens. We have used a combination of anaerobic native liquid chromatography, inductively coupled plasma mass spectrometry, principal-component analysis, and peptide mass fingerprinting to investigate the cytosolic iron distribution in the pathogen Bacillus anthracis. Our studies identified three of the major iron pools as being associated with the electron transfer protein ferredoxin, the miniferritin Dps2, and the superoxide dismutase (SOD) enzymes SodA1 and SodA2. Although both SOD isozymes were predicted to utilize manganese cofactors, quantification of the metal ions associated with SodA1 and SodA2 in cell extracts established that SodA1 is associated with both manganese and iron, whereas SodA2 is bound exclusively to iron in vivo. These data were confirmed by in vitro assays using recombinant protein preparations, showing that SodA2 is active with an iron cofactor, while SodA1 is cambialistic, i.e., active with manganese or iron. Furthermore, we observe that B. anthracis cells exposed to superoxide stress increase their total iron content more than 2-fold over 60 min, while the manganese and zinc contents are unaffected. Notably, the acquired iron is not localized to the three identified cytosolic iron pools.

Comparative analysis of the responses of related pathogenic and environmental bacteria to oxidative stress.

Bacillus anthracis, the causative agent of anthrax, is exposed to host-mediated antibacterial activities, such as reactive oxygen species (ROS), during the early stages of its disease process. The ability to resist these host-mediated stresses is an essential characteristic of a successful pathogen while it is generally assumed that non-pathogenic environmental bacteria succumb to these antimicrobial activities. In order to gain insights into the underlying mechanisms that pathogens use to resist host-mediated oxidative stress, we have compared the oxidative stress responses of B. anthracis and Bacillus subtilis, a well-studied environmental bacterium. Among the four putative catalases encoded by B. anthracis we identified KatB as the main vegetative catalase. Comparative analysis of catalase production in B. anthracis and B. subtilis in response to superoxide and peroxide stress reveals different expression profiles, even though both are regulated by the PerR repressor, which senses and responds to peroxide stress. A B. anthracis perR deletion mutant exhibits enhanced KatB activity and is hyper-resistant to peroxide stress. Superoxide dismutase A1 (SodA1) is the main contributor to the intracellular superoxide dismutase activity in vegetative cells and the gene encoding this enzyme is constitutively expressed. Although aspects of the ROS detoxifying systems of B. anthracis and B. subtilis are similar, their responses to superoxide stress are different. The observed differences are likely to reflect adaptations to specific environmental niches.

The ins and outs of Bacillus proteases: activities, functions and commercial significance.

Because the majority of bacterial species divide by binary fission, and do not have distinguishable somatic and germline cells, they could be considered to be immortal. However, bacteria 'age' due to damage to vital cell components such as DNA and proteins. DNA damage can often be repaired using efficient DNA repair mechanisms. However, many proteins have a functional 'shelf life'; some are short lived, while others are relatively stable. Specific degradation processes are built into the life span of proteins whose activities are required to fulfil a specific function during a prescribed period of time (e.g. cell cycle, differentiation process, stress response). In addition, proteins that are irreparably damaged or that have come to the end of their functional life span need to be removed by quality control proteases. Other proteases are involved in performing a variety of specific functions that can be broadly divided into three categories: processing, regulation and feeding. This review presents a systematic account of the proteases of Bacillus subtilis and their activities. It reviews the proteases found in, or associated with, the cytoplasm, the cell membrane, the cell wall and the external milieu. Where known, the impacts of the deletion of particular proteases are discussed, particularly in relation to industrial applications.

Combined proteomic and transcriptomic analysis of the response of Bacillus anthracis to oxidative stress.

The endospore-forming Gram-positive pathogen Bacillus anthracis is responsible for the usually fatal disease, inhalational anthrax. The success of this pathogen is dependent on its ability to subvert elements of the innate immune system of its animal hosts. B. anthracis spores, which are the main infective agent, are engulfed and germinate in patrolling alveolar macrophages. In order for the infection to progress, the resulting vegetative cells must resist the antimicrobial oxidative burst mounted by the host NADPH oxidase complex. The response of B. anthracis to this and other macrophage-related stresses is therefore of major importance to the success of this pathogen, and consequently we have analysed the superoxide and peroxide stress stimulons of B. anthracis strain UM23C1-2 by means of a combined transcriptomics and proteomics approach. The results show distinct patterns of expression in response to paraquat (endogenous superoxide) and hydrogen peroxide stress. While the main response to paraquat is the induction of iron uptake pathways, the response to peroxide predominantly involves the induction of protection and repair mechanisms. Comparisons between the responses of B. anthracis and related soil bacterium, B. subtilis, reveal differences that are likely to be relevant to their respective habitats.

SmaI restriction site-based multiplex PCR for typing of hospital- and community-acquired Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important nosocomial pathogen, and morbidity and mortality rates associated with this pathogen have increased markedly in recent years. MRSA strains are generally resistant to several classes of antibiotics and are therefore difficult and costly to treat. A major issue is to identify the sources of MRSA infections and to monitor their epidemic spread. In this study, we report the development of a typing technique for S. aureus, based on single-nucleotide polymorphism (SNP) variations in and around SmaI-restriction sites (CCCGGG). An assessment of the SmaI restriction site-based multiplex PCR (SmaI-multiplex PCR) typing (SMT) with respect to pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) revealed a high level of concordance in the clustering of the test strains. The SmaI-multiplex PCR was found to be more discriminatory than MLST/staphylococcal cassette chromosome mec (SCCmec) typing but less discriminatory than PFGE. SMT can provide real-time information for the investigation of ongoing S. aureus hospital outbreaks. SMT meets the criteria of a practical typing method: it is simple, reproducible, and highly discriminatory and does not require expensive equipment or specialist expertise. Consequently, SmaI-multiplex PCR has the potential to be used in routine clinical microbiology laboratories.

In Vitro Oxidative Crosslinking of Recombinant Barnacle Cyprid Cement Gland Proteins.

Barnacle adhesion is a focus for fouling-control technologies as well as the development of bioinspired adhesives, although the mechanisms remain very poorly understood. The barnacle cypris larva is responsible for surface colonisation. Cyprids release cement from paired glands that contain proteins, carbohydrates and lipids, although further compositional details are scant. Several genes coding for cement gland-specific proteins were identified, but only one of these showed database homology. This was a lysyl oxidase-like protein (lcp_LOX). LOX-like enzymes have been previously identified in the proteome of adult barnacle cement secretory tissue. We attempted to produce recombinant LOX in E. coli, in order to identify its role in cyprid cement polymerisation. We also produced two other cement gland proteins (lcp3_36k_3B8 and lcp2_57k_2F5). lcp2_57k_2F5 contained 56 lysine residues and constituted a plausible substrate for LOX. While significant quantities of soluble lcp3_36k_3B8 and lcp2_57k_2F5 were produced in E. coli, production of stably soluble lcp_LOX failed. A commercially sourced human LOX catalysed the crosslinking of lcp2_57k_2F5 into putative dimers and trimers, and this reaction was inhibited by lcp3_36k_3B8. Inhibition of the lcp_LOX:lcp2_57k_2F5 reaction by lcp3_36k_3B8 appeared to be substrate specific, with no inhibitory effect on the oxidation of cadaverine by LOX. The results demonstrate a possible curing mechanism for barnacle cyprid cement and, thus, provide a basis for a more complete understanding of larval adhesion for targeted control of marine biofouling and adhesives for niche applications.

Heterologous protein secretion by bacillus species from the cradle to the grave.

The Gram-positive bacterium Bacillus subtilis and some of its close relatives are widely used for the industrial production of enzymes for the detergents, food, and beverage industries. The choice of these organisms is based almost exclusively on the high capacity of their secretion systems that are, under the right conditions, able to secrete proteins at grams per liter concentrations. In contrast, there are relatively few examples of Bacillus species being used for the cytoplasmic production of proteins. The range of proteins that are capable of high-level production and secretion is limited by a combination of characteristics of both the target protein and the host bacterium. The secretion pathway includes checkpoints that are designed to validate the authenticity of pathway substrates. Although many of these checkpoints are known, only some can be overcome by reengineering the host. As a result, the yield of heterologous protein production is extremely variable. In this review, we consider the Bacillus protein secretion pathway from the synthesis of the target protein (cradle) to its emergence at the outer surface of the complex cell wall (grave), and discuss the roles of the various checkpoints both with respect to the target protein and their role on cell homeostasis.

Development of vaccines at the time of COVID-19.

In December 2019, a working group of the European Academy of Microbiology assembled to discuss various aspects of vaccines and vaccinations. The meeting was organised by Jörg Hacker and Eliora Z. Ron and took place in the offices of the Leopoldina (German National Academy of Sciences Leopoldina). Several important issues were addressed and a major part of the discussion focused on the need to develop new vaccines, especially to protect against pathogens that constitute a pandemic threat. Following the rapid and unpredicted spread of COVID-19 in the first seven months of 2020, the need to develop vaccines for pandemic viruses rapidly has been clearly established. Thus, this paper will concentrate on points that were highlighted by the recent COVID-19 pandemic and lessons learnt therefrom.

Bacillus protein secretion: an unfolding story.

Bacillus subtilis and its close relatives are widely used for the production of enzymes for the detergent, food and beverage industries. These organisms not only produce an appropriate range of enzymes but also have the capacity to secrete them into the culture medium at high concentrations. Purification from the culture medium rather than from the cytoplasm considerably reduces downstream processing costs. In recent years, considerable effort has been aimed at developing B. subtilis as a host for the production of heterologous proteins. The folded state of the target protein at various stages of the secretion pathway has proved to be important.

Changes in the absorption and scattering properties in the near-infrared region during the growth of Bacillus subtilis in liquid culture.

Multiple scattering of light by cells poses a significant challenge in the development of near-infrared-based methodologies to reliably extract chemical and physical information contained in the spectra collected during the bacterial growth cycle. The extent of information that can be obtained from NIR spectra could, in principle, be vastly improved if the scattering and absorption effects can be effectively separated. This study focuses on the methodology for extracting the bulk optical properties over the course of the bacterial growth cycle and investigates the nature and extent of changes in the optical properties with time. By inverting the radiative transfer equation (RTE) using three measurements, total diffuse reflectance, total diffuse transmittance, and collimated transmittance, the bulk absorption coefficient (microa), the bulk scattering coefficient (micros), and the anisotropy factor (g) are extracted and their changes during the course of the growth cycle are investigated. In this study, a simple bacterial growth system consisting of Bacillus subtilis growing in an aqueous solution (minimum medium) was investigated. The changes in the optical properties of this system during bacterial growth, stationary, and decline phases were investigated by inverting the measurements using the adding-doubling method to solve the RTE in the wavelength region of 950-1850 nm. This study shows that during growth in liquid culture, the absorption and scattering property changes can be consistently extracted from measurements under multiple light scattering conditions. The estimation of the anisotropy factor was not reliable beyond 1200 nm at low bacterial cell counts, but reliability increased with increasing biomass concentration. At all stages in the growth cycle, the anisotropy factor could not be reliably extracted in the first overtone region. However, this does not appear to adversely affect the estimation of the absorption and scattering coefficients.

Impact of mass migrations on the clonal variation of clinical Staphylococcus aureus strains isolated from the Western region of Saudi Arabia.

OBJECTIVES: A rapid molecular typing system was used to determine the impact of mass migration on the clonal variation of Staphylococcus aureus isolates recovered from King Abdulaziz University Hospital (KAUH) Jeddah, in the western region of Saudi Arabia. This region experiences an annual influx of millions of pilgrims. METHODS: SmaI-multiplex PCR typing (SMT) was used for the initial analysis of strains and the resulting data subsequently supported by Multi-Locus Sequence Typing (MLST). RESULTS: A total of 89 S. aureus isolates were SMT typed and revealed a high degree of genetic variation, with 40 SMT profiles detected among the isolates. Representatives of all forty SMT types were subsequently analysed by MLST, identifying 26 sequence types. A novel sequence type (ST), named ST3303, was identified in two methicillin-sensitive S. aureus (MSSA) isolates. MSSA strains exhibited more diversity than methicillin-resistant S. aureus (MRSA) strains, with community acquired MSSA and MRSA strains reaching alarmingly high levels. CONCLUSION: The relatively high degree of genetic diversity found among S. aureus isolates of single hospital was attributed to the fact that Jeddah is the principal gateway to Mecca, visited each year by millions of pilgrims from many countries. The observed diversity clearly reflects the impact of such mass migrations in the rapid dissemination of strains world-wide. Our findings suggest the importance of surveillance programmes in locations affected by mass migrations, both to monitor their impact on endemic strains and for the detection of pandemic strains. SMT provides a cost-effective and sensitive typing method for achieving this objective.

Secondary metabolite production and the safety of industrially important members of the Bacillus subtilis group.

Members of the 'Bacillus subtilis group' include some of the most commercially important bacteria, used for the production of a wide range of industrial enzymes and fine biochemicals. Increasingly, group members have been developed for use as animal feed enhancers and antifungal biocontrol agents. The group has long been recognised to produce a range of secondary metabolites and, despite their long history of safe usage, this has resulted in an increased focus on their safety. Traditional methods used to detect the production of secondary metabolites and other potentially harmful compounds have relied on phenotypic tests. Such approaches are time consuming and, in some cases, lack specificity. Nowadays, accessibility to genome data and associated bioinformatical tools provides a powerful means for identifying gene clusters associated with the synthesis of secondary metabolites. This review focuses primarily on well-characterised strains of B. subtilis and B. licheniformis and their synthesis of non-ribosomally synthesised peptides and polyketides. Where known, the activities and toxicities of their secondary metabolites are discussed, together with the limitations of assays currently used to assess their toxicity. Finally, the regulatory framework under which such strains are authorised for use in the production of food and feed enzymes is also reviewed.

Bacillus subtilis, the model Gram-positive bacterium: 20 years of annotation refinement.

Genome annotation is, nowadays, performed via automatic pipelines that cannot discriminate between right and wrong annotations. Given their importance in increasing the accuracy of the genome annotations of other organisms, it is critical that the annotations of model organisms reflect the current annotation gold standard. The genome of Bacillus subtilis strain 168 was sequenced twenty years ago. Using a combination of inductive, deductive and abductive reasoning, we present a unique, manually curated annotation, essentially based on experimental data. This reveals how this bacterium lives in a plant niche, while carrying a paleome operating system common to Firmicutes and Tenericutes. Dozens of new genomic objects and an extensive literature survey have been included for the sequence available at the INSDC (AccNum AL009126.3). We also propose an extension to Demerec's nomenclature rules that will help investigators connect to this type of curated annotation via the use of common gene names.

Co-optimization of sponge-core bioreactors for removing total nitrogen and antibiotic resistance genes from domestic wastewater.

Inadequate sanitation can lead to the spread of infectious diseases and antimicrobial resistance (AMR) via contaminated water. Unfortunately, wastewater treatment is not universal in many developing and emerging countries, especially in rural and peri-urban locations that are remote from central sewers. As such, small-scale, more sustainable treatment options are needed, such as aerobic-Denitrifying Downflow Hanging Sponge (DDHS) bioreactors. In this study, DDHS reactors were assessed for such applications, and achieved over 79% and 84% removal of Chemical Oxygen Demand and Ammonium, respectively, and up to 71% removal of Total Nitrogen (TN) from domestic wastes. Elevated TN removals were achieved via bypassing a fraction of raw wastewater around the top layer of the DDHS system to promote denitrification. However, it was not known how this bypass impacts AMR gene (ARG) and mobile genetic element (MGE) levels in treated effluents. High-throughput qPCR was used to quantify ARG and MGE levels in DDHS bioreactors as a function of percent bypass (0, 10, 20 and 30% by volume). All systems obtained over 90% ARG reduction, although effluent ARG and TN levels differed among bypass regimes, with co-optimal reductions occurring at ~20% bypass. ARG removal paralleled bacterial removal rate, although effluent bacteria tended to have greater genetic plasticity based on higher apparent MGE levels per cell. Overall, TN removal increased and ARG removal decreased with increasing bypass, therefore co-optimization is needed in each DDHS application to achieve locally targeted TN and AMR effluent levels.