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 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.

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.

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.

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.

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.

Exploring the Nonconserved Sequence Space of Synthetic Expression Modules in Bacillus subtilis.

Increasing protein expression levels is a key step in the commercial production of enzymes. Predicting promoter activity and translation initiation efficiency based solely on consensus sequences have so far met with mixed results. Here, we addressed this challenge using a "brute-force" approach by designing and synthesizing a large combinatorial library comprising ∼12 000 unique synthetic expression modules (SEMs) for Bacillus subtilis. Using GFP fluorescence as a reporter of gene expression, we obtained a dynamic expression range that spanned 5 orders of magnitude, as well as a maximal 13-fold increase in expression compared with that of the already strong veg expression module. Analyses of the synthetic modules indicated that sequences at the 5'-end of the mRNA were the most important contributing factor to the differences in expression levels, presumably by preventing formation of strong secondary mRNA structures that affect translation initiation. When the gfp coding region was replaced by the coding region of the xynA gene, encoding the industrially relevant B. subtilis xylanase enzyme, only a 3-fold improvement in xylanase production was observed. Moreover, the correlation between GFP and xylanase expression levels was weak. This suggests that the differences in expression levels between the gfp and xynA constructs were due to differences in 5'-end mRNA folding and consequential differences in the rates of translation initiation. Our data show that the use of large libraries of SEMs, in combination with high-throughput technologies, is a powerful approach to improve the production of a specific protein, but that the outcome cannot necessarily be extrapolated to other proteins.

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.

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.

Extracellular Self-Assembly of Functional and Tunable Protein Conjugates from Bacillus subtilis.

The ability to stably and specifically conjugate recombinant proteins to one another is a powerful approach for engineering multifunctional enzymes, protein therapeutics, and novel biological materials. While many of these applications have been illustrated through in vitro and in vivo intracellular protein conjugation methods, extracellular self-assembly of protein conjugates offers unique advantages: simplifying purification, reducing toxicity and burden, and enabling tunability. Exploiting the recently described SpyTag-SpyCatcher system, we describe here how enzymes and structural proteins can be genetically encoded to covalently conjugate in culture media following programmable secretion from Bacillus subtilis. Using this approach, we demonstrate how self-conjugation of a secreted industrial enzyme, XynA, dramatically increases its resilience to boiling, and we show that cellular consortia can be engineered to self-assemble functional protein-protein conjugates with tunable composition. This novel genetically encoded modular system provides a flexible strategy for protein conjugation harnessing the substantial advantages of extracellular self-assembly.

Effect of Genome Position on Heterologous Gene Expression in Bacillus subtilis: An Unbiased Analysis.

A fixed gene copy number is important for the in silico construction of engineered synthetic networks. However, the copy number of integrated genes depends on their genomic location. This gene dosage effect is rarely addressed in synthetic biology. Two studies in Escherichia coli presented conflicting data on the impact of gene dosage. Here, we investigate how genome location and gene orientation influences expression in Bacillus subtilis. An important difference with the E. coli studies is that we used an unbiased genome integration approach mediated by random transposon insertion. We found that there is a strong gene dosage effect in fast growing B. subtilis cells, which can amount to a 5-fold difference in gene expression. In contrast, gene orientation with respect to DNA replication direction does not influence gene expression. Our study shows that gene dosage should be taken into account when designing synthetic circuits in B. subtilis and presumably other bacteria.

The Identification of Intrinsic Chloramphenicol and Tetracycline Resistance Genes in Members of the Bacillus cereus Group (sensu lato).

Bacillus toyonensis strain BCT-7112T (NCIMB 14858T) has been widely used as an additive in animal nutrition for more than 30 years without reports of adverse toxigenic effects. However, this strain is resistant to chloramphenicol and tetracycline and it is generally considered inadvisable to introduce into the food chain resistance determinants capable of being transferred to other bacterial strains, thereby adding to the pool of such determinants in the gastro-enteric systems of livestock species. We therefore characterized the resistance phenotypes of this strain and its close relatives to determine whether they were of recent origin, and therefore likely to be transmissible. To this end we identified the genes responsible for chloramphenicol (catQ) and tetracycline (tetM) resistance and confirmed the presence of homologs in other members of the B. toyonensis taxonomic unit. Unexpectedly, closely related strains encoding these genes did not exhibit chloramphenicol and tetracycline resistance phenotypes. To understand the differences in the behaviors, we cloned and expressed the genes, together with their upstream regulatory regions, into Bacillus subtilis. The data showed that the genes encoded functional proteins, but were expressed inefficiently from their native promoters. B. toyonensis is a taxonomic unit member of the Bacillus cereus group (sensu lato). We therefore extended the analysis to determine the extent to which homologous chloramphenicol and tetracycline resistance genes were present in other species within this group. This analysis revealed that homologous genes were present in nearly all representative species within the B. cereus group (sensu lato). The absence of known transposition elements and the observations that they are found at the same genomic locations, indicates that these chloramphenicol and tetracycline resistance genes are of ancient origin and intrinsic to this taxonomic group, rather than recent acquisitions. In this context we discuss definitions of what are and are not intrinsic genes, an issue that is of fundamental importance to both Regulatory Authorities, and the animal feed and related industries.

Binase-like guanyl-preferring ribonucleases are new members of Bacillus PhoP regulon.

Extracellular low-molecular weight guanyl-preferring ribonucleases (LMW RNases) of Bacillus sp. comprise a group of hydrolytic enzymes that share highly similar structural and catalytic characteristics with barnase, a ribonuclease from Bacillus amyloliquefaciens, and binase, a ribonuclease from Bacillus intermedius. Although the physical-chemical and catalytic properties of Bacillus guanyl-preferring ribonucleases are very similar, there is considerably more variation in the environmental conditions that lead to the induction of the genes encoding these RNases. Based on structural differences of their genes the guanyl-preferring ribonucleases have been sub-divided into binase-like and barnase-like groups. Here we show the ability of the key regulator of phosphate deficiency response, PhoP, to direct the transcription of the binase-like RNases but not barnase-like RNases. These results, together with our demonstration that binase-like RNases are induced in response to phosphate starvation, allow us to categorise this group of ribonucleases as new members of Bacillus PhoP regulon. In contrast, the barnase-like ribonucleases are relatively insensitive to the phosphate concentration and the environmental conditions that are responsible for their induction, and the regulatory elements involved, are currently unknown.

A distributed computational search strategy for the identification of diagnostics targets: application to finding aptamer targets for methicillin-resistant staphylococci.

The rapid and cost-effective identification of bacterial species is crucial, especially for clinical diagnosis and treatment. Peptide aptamers have been shown to be valuable for use as a component of novel, direct detection methods. These small peptides have a number of advantages over antibodies, including greater specificity and longer shelf life. These properties facilitate their use as the detector components of biosensor devices. However, the identification of suitable aptamer targets for particular groups of organisms is challenging. We present a semi-automated processing pipeline for the identification of candidate aptamer targets from whole bacterial genome sequences. The pipeline can be configured to search for protein sequence fragments that uniquely identify a set of strains of interest. The system is also capable of identifying additional organisms that may be of interest due to their possession of protein fragments in common with the initial set. Through the use of Cloud computing technology and distributed databases, our system is capable of scaling with the rapidly growing genome repositories, and consequently of keeping the resulting data sets up-to-date. The system described is also more generically applicable to the discovery of specific targets for other diagnostic approaches such as DNA probes, PCR primers and antibodies.

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.

Microbase2.0: a generic framework for computationally intensive bioinformatics workflows in the cloud.

As bioinformatics datasets grow ever larger, and analyses become increasingly complex, there is a need for data handling infrastructures to keep pace with developing technology. One solution is to apply Grid and Cloud technologies to address the computational requirements of analysing high throughput datasets. We present an approach for writing new, or wrapping existing applications, and a reference implementation of a framework, Microbase2.0, for executing those applications using Grid and Cloud technologies. We used Microbase2.0 to develop an automated Cloud-based bioinformatics workflow executing simultaneously on two different Amazon EC2 data centres and the Newcastle University Condor Grid. Several CPU years' worth of computational work was performed by this system in less than two months. The workflow produced a detailed dataset characterising the cellular localisation of 3,021,490 proteins from 867 taxa, including bacteria, archaea and unicellular eukaryotes. Microbase2.0 is freely available from http://www.microbase.org.uk/.