Ancestry of RNA/RNA interaction regions within segmented ribosomes.

The ribosome is the universally conserved ribozyme that translates DNA coded instructions into proteins with the assistance of other RNA molecules, including transfer and messenger RNAs. Of particular interest is the segmentation phenomena, which is found in trypanosomatids and other protists. In these organisms, the large subunit ribosomal RNA is assembled from multiple smaller RNAs. This phenomenon posits several challenges to the folding and stabilization of such ribosomes to retain functionality and efficiency. In earlier studies, RNA/protein interactions were suggested to fully compensate for the fragmentation. Recently, several conserved RNA/RNA interaction regions were described in the cryo-EM structures of segmented ribosomes from trypanosomatids. These regions also seemed to aid in the folding and stabilization of such ribosomes, even before the ribosomal proteins start their association. In the present study, the existence of conserved RNA/RNA interaction regions shared between trypanosomatid and Euglena gracilis segmented ribosomes was confirmed, despite differences in segmentation patterns. Analysis of the crystallographic structures of unsegmented ribosomes from other Eukaryotes, Bacteria, and Archaea allowed us to estimate the relative age of highly conserved RNA/RNA interaction regions. These results strongly suggest that common interaction regions likely date far back into the ribosomes of the last common ancestor. Results also revealed that single hydrogen bonds are overwhelmingly facilitated by the 2'OH, a distinctive RNA feature. This supports the notion that RNA predates DNA and places some constraints on alternative nucleic acids proposals.

How to build a protoribosome: structural insights from the first protoribosome constructs that have proven to be catalytically active.

The modern ribosome catalyzes all coded protein synthesis in extant organisms. It is likely that its core structure is a direct descendant from the ribosome present in the last common ancestor (LCA). Hence, its earliest origins likely predate the LCA and therefore date further back in time. Of special interest is the pseudosymmetrical region (SymR) that lies deep within the large subunit (LSU) where the peptidyl transfer reaction takes place. It was previously proposed that two RNA oligomers, representing the P- and A-regions of extant ribosomes dimerized to create a pore-like structure, which hosted the necessary properties that facilitate peptide bond formation. However, recent experimental studies show that this may not be the case. Instead, several RNA constructs derived exclusively from the P-region were shown to form a homodimer capable of peptide bond synthesis. Of special interest will be the origin issues because the homodimer would have allowed a pre-LCA ribosome that was significantly smaller than previously proposed. For the A-region, the immediate issue will likely be its origin and whether it enhances ribosome performance. Here, we reanalyze the RNA/RNA interaction regions that most likely lead to SymR formation in light of these recent findings. Further, it has been suggested that the ability of these RNA constructs to dimerize and enhance peptide bond formation is sequence-dependent. We have analyzed the implications of sequence variations as parts of functional and nonfunctional constructs.

Ribosomal Protein Cluster Organization in Asgard Archaea.

It has been proposed that the superphylum of Asgard Archaea may represent a historical link between the Archaea and Eukarya. Following the discovery of the Archaea, it was soon appreciated that archaeal ribosomes were more similar to those of Eukarya rather than Bacteria. Coupled with other eukaryotic-like features, it has been suggested that the Asgard Archaea may be directly linked to eukaryotes. However, the genomes of Bacteria and non-Asgard Archaea generally organize ribosome-related genes into clusters that likely function as operons. In contrast, eukaryotes typically do not employ an operon strategy. To gain further insight into conservation of the r-protein genes, the genome order of conserved ribosomal protein (r-protein) coding genes was identified in 17 Asgard genomes (thirteen complete genomes and four genomes with less than 20 contigs) and compared with those found previously in non-Asgard archaeal and bacterial genomes. A universal core of two clusters of 14 and 4 cooccurring r-proteins, respectively, was identified in both the Asgard and non-Asgard Archaea. The equivalent genes in the E. coli version of the cluster are found in the S10 and spc operons. The large cluster of 14 r-protein genes (uS19-uL22-uS3-uL29-uS17 from the S10 operon and uL14-uL24-uL5-uS14-uS8-uL6-uL18-uS5-uL30-uL15 from the spc operon) occurs as a complete set in the genomes of thirteen Asgard genomes (five Lokiarchaeotes, three Heimdallarchaeotes, one Odinarchaeote, and four Thorarchaeotes). Four less conserved clusters with partial bacterial equivalents were found in the Asgard. These were the L30e (str operon in Bacteria) cluster, the L18e (alpha operon in Bacteria) cluster, the S24e-S27ae-rpoE1 cluster, and the L31e, L12..L1 cluster. Finally, a new cluster referred to as L7ae was identified. In many cases, r-protein gene clusters/operons are less conserved in their organization in the Asgard group than in other Archaea. If this is generally true for nonribosomal gene clusters, the results may have implications for the history of genome organization. In particular, there may have been an early transition to or from the operon approach to genome organization. Other nonribosomal cellular features may support different relationships. For this reason, it may be important to consider ribosome features separately.

Nonstandard RNA/RNA interactions likely enhance folding and stability of segmented ribosomes.

The ribosome is the molecular factory that catalyzes all coded protein synthesis in extant organisms. Eukaryotic ribosomes are typically assembled out of four rRNAs; namely, 5S, 5.8S, 18S, and 28S. However, the 28S rRNA of some trypanosomatid organisms has been found to be segmented into six independent rRNAs of different sizes. The two largest segments have multiple sites where they jointly form stems comprised of standard base pairs that can hold them together. However, such regions of interaction are not observed among the four smaller RNAs. Early reports suggested that trypanosomatid segmented ribosome assembly was essentially achieved thanks to their association with rProteins. However, examination of cryo-EM ribosomal structures from Trypanosoma brucei, Leishmania donovani, and Trypanosoma cruzi reveals several long-range nonstandard RNA/RNA interactions. Most of these interactions are clusters of individual hydrogen bonds and so are not readily predictable. However, taken as a whole, they represent significant stabilizing energy that likely facilitates rRNA assembly and the overall stability of the segmented ribosomes. In the context of origin of life studies, the current results provide a better understanding of the true nature of RNA sequence space and what might be possible without an RNA replicase.

Net Charges of the Ribosomal Proteins of the S10 and spc Clusters of Halophiles Are Inversely Related to the Degree of Halotolerance.

Net positive charge(s) on ribosomal proteins (r-proteins) have been reported to influence the assembly and folding of ribosomes. A high percentage of r-proteins from extremely halophilic archaea are known to be acidic or even negatively charged. Those proteins that remain positively charged are typically far less positively charged. Here, the analysis is extended to non-archaeal halophilic bacteria, eukaryotes, and halotolerant archaea. The net charges (pH 7.4) of the r-proteins that comprise the S10-spc operon/cluster from individual microbial and eukaryotic genomes were estimated and intercompared. It was observed that, as a general rule, the net charges of individual proteins remained mostly basic as the salt tolerance of the bacterial strains increased from 5 to 15%. The most striking exceptions were the extremely halophilic bacterial strains, Salinibacter ruber SD01, Acetohalobium arabaticum DSM 5501 and Selenihalanaerobacter shriftii ATCC BAA-73, which are reported to require a minimum of 18% to 21% salt for their growth. All three strains have higher numbers of acidic S10-spc cluster r-proteins than what is seen in the moderate halophiles or the halotolerant strains. Of the individual proteins, only uL2 never became acidic. uS14 and uL16 also seldom became acidic. The net negative charges on several of the S10-spc cluster r-proteins are a feature generally shared by all extremely halophilic archaea and bacteria. The S10-spc cluster r-proteins of halophilic fungi and algae (eukaryotes) were exceptions: these were positively charged despite the halophilicity of the organisms. IMPORTANCE The net charges (at pH 7.4) of the ribosomal proteins (r-proteins) that comprise the S10-spc cluster show an inverse relationship with the halophilicity/halotolerance levels in both bacteria and archaea. In non-halophilic bacteria, the S10-spc cluster r-proteins are generally basic (positively charged), while the rest of the proteomes in these strains are generally acidic. On the other hand, the whole proteomes of the extremely halophilic strains are overall negatively charged, including the S10-spc cluster r-proteins. Given that the distribution of charged residues in the ribosome exit tunnel influences cotranslational folding, the contrasting charges observed in the S10-spc cluster r-proteins have potential implications for the rate of passage of these proteins through the ribosomal exit tunnel. Furthermore, the universal protein uL2, which lies in the oldest part of the ribosome, is always positively charged irrespective of the strain/organism it belongs to. This has implications for its role in the prebiotic context.

The Peptidyl Transferase Center: a Window to the Past.

In his 2001 article, "Translation: in retrospect and prospect," the late Carl Woese made a prescient observation that there was a need for the then-current view of translation to be "reformulated to become an all-embracing perspective about which 21st century Biology can develop" (RNA 7:1055-1067, 2001, https://doi.org/10.1017/s1355838201010615). The quest to decipher the origins of life and the road to the genetic code are both inextricably linked with the history of the ribosome. After over 60 years of research, significant progress in our understanding of how ribosomes work has been made. Particularly attractive is a model in which the ribosome may facilitate an ∼180° rotation of the CCA end of the tRNA from the A-site to the P-site while the acceptor stem of the tRNA would then undergo a translation from the A-site to the P-site. However, the central question of how the ribosome originated remains unresolved. Along the path from a primitive RNA world or an RNA-peptide world to a proto-ribosome world, the advent of the peptidyl transferase activity would have been a seminal event. This functionality is now housed within a local region of the large-subunit (LSU) rRNA, namely, the peptidyl transferase center (PTC). The PTC is responsible for peptide bond formation during protein synthesis and is usually considered to be the oldest part of the modern ribosome. What is frequently overlooked is that by examining the origins of the PTC itself, one is likely going back even further in time. In this regard, it has been proposed that the modern PTC originated from the association of two smaller RNAs that were once independent and now comprise a pseudosymmetric region in the modern PTC. Could such an association have survived? Recent studies have shown that the extant PTC is largely depleted of ribosomal protein interactions. It is other elements like metallic ion coordination and nonstandard base/base interactions that would have had to stabilize the association of RNAs. Here, we present a detailed review of the literature focused on the nature of the extant PTC and its proposed ancestor, the proto-ribosome.

In Vivo Production of Small Recombinant RNAs Embedded in 5S rRNA-Derived Protective Scaffold.

Preparative synthesis of RNA is a challenging task that is usually accomplished by either chemical or enzymatic polymerization of ribonucleotides in vitro. Herein, we describe an alternative approach in which RNAs of interest are expressed as a fusion with a 5S rRNA-derived scaffold. The scaffold provides protection against cellular ribonucleases resulting in cellular accumulations comparable to those of regular ribosomal RNAs. After isolation of the chimeric RNA from the cells, the scaffold can be removed, if necessary, by deoxyribozyme-catalyzed cleavage followed by preparative electrophoretic separation of the reaction products. The protocol is designed for sustained production of high quality RNA on the milligram scale.

Expansion segments in bacterial and archaeal 5S ribosomal RNAs.

The large ribosomal RNAs of eukaryotes frequently contain expansion sequences that add to the size of the rRNAs but do not affect their overall structural layout and are compatible with major ribosomal function as an mRNA translation machine. The expansion of prokaryotic ribosomal RNAs is much less explored. In order to obtain more insight into the structural variability of these conserved molecules, we herein report the results of a comprehensive search for the expansion sequences in prokaryotic 5S rRNAs. Overall, 89 expanded 5S rRNAs of 15 structural types were identified in 15 archaeal and 36 bacterial genomes. Expansion segments ranging in length from 13 to 109 residues were found to be distributed among 17 insertion sites. The strains harboring the expanded 5S rRNAs belong to the bacterial orders Clostridiales, Halanaerobiales, Thermoanaerobacterales, and Alteromonadales as well as the archael order Halobacterales When several copies of a 5S rRNA gene are present in a genome, the expanded versions may coexist with normal 5S rRNA genes. The insertion sequences are typically capable of forming extended helices, which do not seemingly interfere with folding of the conserved core. The expanded 5S rRNAs have largely been overlooked in 5S rRNA databases.

Further Characterization of the Pseudo-Symmetrical Ribosomal Region.

The peptidyl transferase center of the modern ribosome has been found to encompass an area of twofold pseudosymmetry (SymR). This observation strongly suggests that the very core of the ribosome arose from a dimerization event between two modest-sized RNAs. It was previously shown that at least four non-standard interactions exist between the two halves of SymR. Herein, we verify that the structure of the SymR is highly conserved with respect to both ribosome transition state and phylogenetic diversity. These comparisons also reveal two additional sites of interaction between the two halves of SymR and refine our understanding of the previously known interactions. In addition, the possible role that magnesium may have in the coordination, stabilization, association, and evolutionary history of the two halves (A-region and P-region) was examined. Together, the results identify a likely site where structural elements and Mg2+ ions may have facilitated the ligation of two aboriginal RNAs into a single unit.

Cryo-electron microscopy visualization of a large insertion in the 5S ribosomal RNA of the extremely halophilic archaeon Halococcus morrhuae.

The extreme halophile Halococcus morrhuae (ATCC® 17082) contains a 108-nucleotide insertion in its 5S rRNA. Large rRNA expansions in Archaea are rare. This one almost doubles the length of the 5S rRNA. In order to understand how such an insertion is accommodated in the ribosome, we obtained a cryo-electron microscopy reconstruction of the native large subunit at subnanometer resolution. The insertion site forms a four-way junction that fully preserves the canonical 5S rRNA structure. Moving away from the junction site, the inserted region is conformationally flexible and does not pack tightly against the large subunit. The high-salt requirement of the H. morrhuae ribosomes for their stability conflicted with the low-salt threshold for cryo-electron microscopy procedures. Despite this obstacle, this is the first cryo-electron microscopy map of Halococcus ribosomes.

Phobos LIFE (Living Interplanetary Flight Experiment).

The Planetary Society's Phobos Living Interplanetary Flight Experiment (Phobos LIFE) flew in the sample return capsule of the Russian Federal Space Agency's Phobos Grunt mission and was to have been a test of one aspect of the hypothesis that life can move between nearby planets within ejected rocks. Although the Phobos Grunt mission failed, we present here the scientific and engineering design and motivation of the Phobos LIFE experiment to assist with the scientific and engineering design of similar future experiments. Phobos LIFE flew selected organisms in a simulated meteoroid. The 34-month voyage would have been the first such test to occur in the high-radiation environment outside the protection of Earth's magnetosphere for more than a few days. The patented Phobos LIFE "biomodule" is an 88 g cylinder consisting of a titanium outer shell, several types of redundant seals, and 31 individual Delrin sample containers. Phobos LIFE contained 10 different organisms, representing all three domains of life, and one soil sample. The organisms are all very well characterized, most with sequenced genomes. Most are extremophiles, and most have flown in low Earth orbit. Upon return from space, the health and characteristics of organisms were to have been compared with controls that remained on Earth and have not yet been opened.

Characterization of the total and viable bacterial and fungal communities associated with the International Space Station surfaces.

BACKGROUND: The International Space Station (ISS) is a closed system inhabited by microorganisms originating from life support systems, cargo, and crew that are exposed to unique selective pressures such as microgravity. To date, mandatory microbial monitoring and observational studies of spacecraft and space stations have been conducted by traditional culture methods, although it is known that many microbes cannot be cultured with standard techniques. To fully appreciate the true number and diversity of microbes that survive in the ISS, molecular and culture-based methods were used to assess microbial communities on ISS surfaces. Samples were taken at eight pre-defined locations during three flight missions spanning 14 months and analyzed upon return to Earth. RESULTS: The cultivable bacterial and fungal population ranged from 104 to 109 CFU/m2 depending on location and consisted of various bacterial (Actinobacteria, Firmicutes, and Proteobacteria) and fungal (Ascomycota and Basidiomycota) phyla. Amplicon sequencing detected more bacterial phyla when compared to the culture-based analyses, but both methods identified similar numbers of fungal phyla. Changes in bacterial and fungal load (by culture and qPCR) were observed over time but not across locations. Bacterial community composition changed over time, but not across locations, while fungal community remained the same between samplings and locations. There were no significant differences in community composition and richness after propidium monoazide sample treatment, suggesting that the analyzed DNA was extracted from intact/viable organisms. Moreover, approximately 46% of intact/viable bacteria and 40% of intact/viable fungi could be cultured. CONCLUSIONS: The results reveal a diverse population of bacteria and fungi on ISS environmental surfaces that changed over time but remained similar between locations. The dominant organisms are associated with the human microbiome and may include opportunistic pathogens. This study provides the first comprehensive catalog of both total and intact/viable bacteria and fungi found on surfaces in closed space systems and can be used to help develop safety measures that meet NASA requirements for deep space human habitation. The results of this study can have significant impact on our understanding of other confined built environments on the Earth such as clean rooms used in the pharmaceutical and medical industries.

Evaluation of Acquired Antibiotic Resistance in Escherichia coli Exposed to Long-Term Low-Shear Modeled Microgravity and Background Antibiotic Exposure.

The long-term response of microbial communities to the microgravity environment of space is not yet fully understood. Of special interest is the possibility that members of these communities may acquire antibiotic resistance. In this study, Escherichia coli cells were grown under low-shear modeled microgravity (LSMMG) conditions for over 1,000 generations (1000G) using chloramphenicol treatment between cycles to prevent contamination. The results were compared with data from an earlier control study done under identical conditions using steam sterilization between cycles rather than chloramphenicol. The sensitivity of the final 1000G-adapted strain to a variety of antibiotics was determined using Vitek analysis. In addition to resistance to chloramphenicol, the adapted strain acquired resistance to cefalotin, cefuroxime, cefuroxime axetil, cefoxitin, and tetracycline. In fact, the resistance to chloramphenicol and cefalotin persisted for over 110 generations despite the removal of both LSMMG conditions and trace antibiotic exposure. Genome sequencing of the adapted strain revealed 22 major changes, including 3 transposon-mediated rearrangements (TMRs). Two TMRs disrupted coding genes (involved in bacterial adhesion), while the third resulted in the deletion of an entire segment (14,314 bp) of the genome, which includes 14 genes involved with motility and chemotaxis. These results are in stark contrast with data from our earlier control study in which cells grown under the identical conditions without antibiotic exposure never acquired antibiotic resistance. Overall, LSMMG does not appear to alter the antibiotic stress resistance seen in microbial ecosystems not exposed to microgravity.IMPORTANCE Stress factors experienced during space include microgravity, sleep deprivation, radiation, isolation, and microbial contamination, all of which can promote immune suppression (1, 2). Under these conditions, the risk of infection from opportunistic pathogens increases significantly, particularly during long-term missions (3). If infection occurs, it is important that the infectious agent should not be antibiotic resistant. Minimizing the occurrence of antibiotic resistance is, therefore, highly desirable. To facilitate this, it is important to better understand the long-term response of bacteria to the microgravity environment. This study demonstrated that the use of antibiotics as a preventive measure could be counterproductive and would likely result in persistent resistance to that antibiotic. In addition, unintended resistance to other antimicrobials might also occur as well as permanent genome changes that might have other unanticipated and undesirable consequences.

Bacillus safensis FO-36b and Bacillus pumilus SAFR-032: a whole genome comparison of two spacecraft assembly facility isolates.

BACKGROUND: Bacillus strains producing highly resistant spores have been isolated from cleanrooms and space craft assembly facilities. Organisms that can survive such conditions merit planetary protection concern and if that resistance can be transferred to other organisms, a health concern too. To further efforts to understand these resistances, the complete genome of Bacillus safensis strain FO-36b, which produces spores resistant to peroxide and radiation was determined. The genome was compared to the complete genome of B. pumilus SAFR-032, and the draft genomes of B. safensis JPL-MERTA-8-2 and the type strain B. pumilus ATCC7061T. Additional comparisons were made to 61 draft genomes that have been mostly identified as strains of B. pumilus or B. safensis. RESULTS: The FO-36b gene order is essentially the same as that in SAFR-032 and other B. pumilus strains. The annotated genome has 3850 open reading frames and 40 noncoding RNAs and riboswitches. Of these, 307 are not shared by SAFR-032, and 65 are also not shared by MERTA and ATCC7061T. The FO-36b genome has ten unique open reading frames and two phage-like regions, homologous to the Bacillus bacteriophage SPP1 and Brevibacillus phage Jimmer1. Differing remnants of the Jimmer1 phage are found in essentially all B. safensis / B. pumilus strains. Seven unique genes are part of these phage elements. Whole Genome Phylogenetic Analysis of the B. pumilus, B. safensis and other Firmicutes genomes, separate them into three distinct clusters. Two clusters are subgroups of B. pumilus while one houses all the B. safensis strains. The Genome-genome distance analysis and a phylogenetic analysis of gyrA sequences corroborated these results. CONCLUSIONS: It is not immediately obvious that the presence or absence of any specific gene or combination of genes is responsible for the variations in resistance seen. It is quite possible that distinctions in gene regulation can alter the expression levels of key proteins thereby changing the organism's resistance properties without gain or loss of a particular gene. What is clear is that phage elements contribute significantly to genome variability. Multiple genome comparison indicates that many strains named as B. pumilus likely belong to the B. safensis group.

Exploration of RNA Sequence Space in the Absence of a Replicase.

It is generally considered that if an RNA World ever existed that it would be driven by an RNA capable of RNA replication. Whether such a catalytic RNA could emerge in an RNA World or not, there would need to be prior routes to increasing complexity in order to produce it. It is hypothesized here that increasing sequence variety, if not complexity, can in fact readily emerge in response to a dynamic equilibrium between synthesis and degradation. A model system in which T4 RNA ligase catalyzes synthesis and Benzonase catalyzes degradation was constructed. An initial 20-mer served as a seed and was subjected to 180 min of simultaneous ligation and degradation. The seed RNA rapidly disappeared and was replaced by an increasing number and variety of both larger and smaller variants. Variants of 40-80 residues were consistently seen, typically representing 2-4% of the unique sequences. In a second experiment with four individual 9-mers, numerous variants were again produced. These included variants of the individual 9-mers as well as sequences that contained sequence segments from two or more 9-mers. In both cases, the RNA products lack large numbers of point mutations but instead incorporate additions and subtractions of fragments of the original RNAs. The system demonstrates that if such equilibrium were established in a prebiotic world it would result in significant exploration of RNA sequence space and likely increased complexity. It remains to be seen if the variety of products produced is affected by the presence of small peptide oligomers.

Draft Genome Sequence of Marinobacter vinifirmus Type Strain FB1.

The gammaproteobacterium Marinobacter vinifirmus is associated with moderately saline environments and is often found in marine ecosystems. Here, we report the draft genome sequence of M. vinifirmus type strain FB1 (3.8 Mbp, 3,588 predicted genes). The presented sequence will improve our understanding of the taxonomy and evolution of the genus Marinobacter.

Draft Genome Sequences from a Novel Clade of Bacillus cereus Sensu Lato Strains, Isolated from the International Space Station.

The draft genome sequences of six Bacillus strains, isolated from the International Space Station and belonging to the Bacillus anthracis-B. cereus-B. thuringiensis group, are presented here. These strains were isolated from the Japanese Experiment Module (one strain), U.S. Harmony Node 2 (three strains), and Russian Segment Zvezda Module (two strains).

The adaptation of Escherichia coli cells grown in simulated microgravity for an extended period is both phenotypic and genomic.

Microorganisms impact spaceflight in a variety of ways. They play a positive role in biological systems, such as waste water treatment but can be problematic through buildups of biofilms that can affect advanced life support. Of special concern is the possibility that during extended missions, the microgravity environment will provide positive selection for undesirable genomic changes. Such changes could affect microbial antibiotic sensitivity and possibly pathogenicity. To evaluate this possibility, Escherichia coli (lac plus) cells were grown for over 1000 generations on Luria Broth medium under low-shear modeled microgravity conditions in a high aspect rotating vessel. This is the first study of its kind to grow bacteria for multiple generations over an extended period under low-shear modeled microgravity. Comparisons were made to a non-adaptive control strain using growth competitions. After 1000 generations, the final low-shear modeled microgravity-adapted strain readily outcompeted the unadapted lac minus strain. A portion of this advantage was maintained when the low-shear modeled microgravity strain was first grown in a shake flask environment for 10, 20, or 30 generations of growth. Genomic sequencing of the 1000 generation strain revealed 16 mutations. Of the five changes affecting codons, none were neutral. It is not clear how significant these mutations are as individual changes or as a group. It is concluded that part of the long-term adaptation to low-shear modeled microgravity is likely genomic. The strain was monitored for acquisition of antibiotic resistance by VITEK analysis throughout the adaptation period. Despite the evidence of genomic adaptation, resistance to a variety of antibiotics was never observed.

Bacillus pumilus SAFR-032 Genome Revisited: Sequence Update and Re-Annotation.

Bacillus pumilus strain SAFR-032 is a non-pathogenic spore-forming bacterium exhibiting an anomalously high persistence in bactericidal environments. In its dormant state, it is capable of withstanding doses of ultraviolet (UV) radiation or hydrogen peroxide, which are lethal for the vast majority of microorganisms. This unusual resistance profile has made SAFR-032 a reference strain for studies of bacterial spore resistance. The complete genome sequence of B. pumilus SAFR-032 was published in 2007 early in the genomics era. Since then, the SAFR-032 strain has frequently been used as a source of genetic/genomic information that was regarded as representative of the entire B. pumilus species group. Recently, our ongoing studies of conservation of gene distribution patterns in the complete genomes of various B. pumilus strains revealed indications of misassembly in the B. pumilus SAFR-032 genome. Synteny-driven local genome resequencing confirmed that the original SAFR-032 sequence contained assembly errors associated with long sequence repeats. The genome sequence was corrected according to the new findings. In addition, a significantly improved annotation is now available. Gene orders were compared and portions of the genome arrangement were found to be similar in a wide spectrum of Bacillus strains.