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.

Effects of Beneficial Mutations in pykF Gene Vary over Time and across Replicate Populations in a Long-Term Experiment with Bacteria.

The fitness effects of mutations can depend on the genetic backgrounds in which they occur and thereby influence future opportunities for evolving populations. In particular, mutations that fix in a population might change the selective benefit of subsequent mutations, giving rise to historical contingency. We examine these effects by focusing on mutations in a key metabolic gene, pykF, that arose independently early in the history of 12 Escherichia coli populations during a long-term evolution experiment. Eight different evolved nonsynonymous mutations conferred similar fitness benefits of ∼10% when transferred into the ancestor, and these benefits were greater than the one conferred by a deletion mutation. In contrast, the same mutations had highly variable fitness effects, ranging from ∼0% to 25%, in evolved clones isolated from the populations at 20,000 generations. Two mutations that were moved into these evolved clones conferred similar fitness effects in a given clone, but different effects between the clones, indicating epistatic interactions between the evolved pykF alleles and the other mutations that had accumulated in each evolved clone. We also measured the fitness effects of six evolved pykF alleles in the same populations in which they had fixed, but at seven time points between 0 and 50,000 generations. Variation in fitness effects was high at intermediate time points, and declined to a low level at 50,000 generations, when the mean fitness effect was lowest. Our results demonstrate the importance of genetic context in determining the fitness effects of different beneficial mutations even within the same gene.

Diminishing-returns epistasis decreases adaptability along an evolutionary trajectory.

Populations evolving in constant environments exhibit declining adaptability. Understanding the basis of this pattern could reveal underlying processes determining the repeatability of evolutionary outcomes. In principle, declining adaptability can be due to a decrease in the effect size of beneficial mutations, a decrease in the rate at which they occur, or some combination of both. By evolving Escherichia coli populations started from different steps along a single evolutionary trajectory, we show that declining adaptability is best explained by a decrease in the size of available beneficial mutations. This pattern reflected the dominant influence of negative genetic interactions that caused new beneficial mutations to confer smaller benefits in fitter genotypes. Genome sequencing revealed that starting genotypes that were more similar to one another did not exhibit greater similarity in terms of new beneficial mutations, supporting the view that epistasis acts globally, having a greater influence on the effect than on the identity of available mutations along an adaptive trajectory. Our findings provide support for a general mechanism that leads to predictable phenotypic evolutionary trajectories.

Benefit of transferred mutations is better predicted by the fitness of recipients than by their ecological or genetic relatedness.

The effect of a mutation depends on its interaction with the genetic background in which it is assessed. Studies in experimental systems have demonstrated that such interactions are common among beneficial mutations and often follow a pattern consistent with declining evolvability of more fit genotypes. However, these studies generally examine the consequences of interactions between a small number of focal mutations. It is not clear, therefore, that findings can be extrapolated to natural populations, where new mutations may be transferred between genetically divergent backgrounds. We build on work that examined interactions between four beneficial mutations selected in a laboratory-evolved population of Escherichia coli to test how they interact with the genomes of diverse natural isolates of the same species. We find that the fitness effect of transferred mutations depends weakly on the genetic and ecological similarity of recipient strains relative to the donor strain in which the mutations were selected. By contrast, mutation effects were strongly inversely correlated to the initial fitness of the recipient strain. That is, there was a pattern of diminishing returns whereby fit strains benefited proportionally less from an added mutation. Our results strengthen the view that the fitness of a strain can be a major determinant of its ability to adapt. They also support a role for barriers of transmission, rather than differential selection of transferred DNA, as an explanation of observed phylogenetically determined patterns of restricted recombination among E. coli strains.

Adaptation of Escherichia coli to glucose promotes evolvability in lactose.

The selective history of a population can influence its subsequent evolution, an effect known as historical contingency. We previously observed that five of six replicate populations that were evolved in a glucose-limited environment for 2000 generations, then switched to lactose for 1000 generations, had higher fitness increases in lactose than populations started directly from the ancestor. To test if selection in glucose systematically increased lactose evolvability, we started 12 replay populations--six from a population subsample and six from a single randomly selected clone--from each of the six glucose-evolved founder populations. These replay populations and 18 ancestral populations were evolved for 1000 generations in a lactose-limited environment. We found that replay populations were initially slightly less fit in lactose than the ancestor, but were more evolvable, in that they increased in fitness at a faster rate and to higher levels. This result indicates that evolution in the glucose environment resulted in genetic changes that increased the potential of genotypes to adapt to lactose. Genome sequencing identified four genes--iclR, nadR, spoT, and rbs--that were mutated in most glucose-evolved clones and are candidates for mediating increased evolvability. Our results demonstrate that short-term selective costs during selection in one environment can lead to changes in evolvability that confer longer term benefits.

Natural variation in the promoter of the gene encoding the Mga regulator alters host-pathogen interactions in group a Streptococcus carrier strains.

Humans commonly carry pathogenic bacteria asymptomatically, but the molecular factors underlying microbial asymptomatic carriage are poorly understood. We previously reported that two epidemiologically unassociated serotype M3 group A Streptococcus (GAS) carrier strains had an identical 12-bp deletion in the promoter of the gene encoding Mga, a global positive gene regulator. Herein, we report on studies designed to test the hypothesis that the identified 12-bp deletion in the mga promoter alters GAS virulence, thereby potentially contributing to the asymptomatic carrier phenotype. Using allelic exchange, we introduced the variant promoter into a serotype M3 invasive strain and the wild-type promoter into an asymptomatic carrier strain. Compared to strains with the wild-type mga promoter, we discovered that strains containing the promoter with the 12-bp deletion produced significantly fewer mga and Mga-regulated gene transcripts. Consistent with decreased mga transcripts, strains containing the variant mga promoter were also significantly less virulent in in vivo and ex vivo models of GAS disease. Further, we provide evidence that the pleiotropic regulator protein CodY binds to the mga promoter and that the 12-bp deletion in the mga promoter reduces CodY-mediated mga transcription. We conclude that the naturally occurring 12-bp deletion in the mga promoter significantly alters the pathogen-host interaction of these asymptomatic carrier strains. Our findings provide new insight into the molecular basis of the carrier state of an important human pathogen.

CcpA forms complexes with CodY and RpoA in Bacillus subtilis.

The Bacillus subtilis catabolite control protein A (CcpA) is a global transcriptional regulator that is controlled by interactions with the phosphoproteins histidine-containing protein (HPr)Ser46P and the catabolite responsive HPr (Crh)Ser46P and with low molecular weight effectors, depending on the availability of preferred carbon sources such as glucose. Distinct point mutations in CcpA abolish the regulation of some but not all target genes, suggesting additional interactions of CcpA. Therefore, in vivo crosslinking and MS were applied to identify CcpA complexes active in repression and activation. To compensate for an excess of promoters only repressed by CcpA, this experiment was accomplished with cells using multiple copies of the activated ackA promoter. Among the identified proteins HPr, RNA polymerase subunits and the global regulator transcriptional pleiotropic repressor (CodY) were observed. Bacterial two-hybrid assays combining each RNA polymerase subunit with CcpA localized CcpA binding at the α-subunit of the RNA polymerase (RpoA). In vivo crosslinking combined with immunoblot analyses revealed CcpA-RpoA complexes in cultures with or without glucose, whereas CcpA-HPr and CcpA-CodY complexes occurred only or predominantly in cultures with glucose. Surface plasmon resonance analyses confirmed the binding of CcpA to the N-terminal domain (αNTD) and C-terminal domain (αCTD) of RpoA, as well as to CodY. Furthermore, interactions of CodY with the αNTD and the αCTD were detected by surface plasmon resonance. The K(D) values of complexes of CcpA or CodY with the αNTD or the αCTD are in the range 5-8 µm. CcpA and CodY form a loose complex with a K(D) of 60 µm. These data were combined to propose a model for a transcription initiation complex at the ackA promoter.

Regulated expression of HPrK/P does not affect carbon catabolite repression of the xyn operon and of rocG in Bacillus subtilis.

HPr kinase/phosphorylase (HPrK/P), a central metabolic regulator in many Gram-positive bacteria, reversibly phosphorylates HPr and Crh, thus controlling their activities as effectors of CcpA predominantly in carbon catabolite repression (CCR). We have placed the constitutively expressed hprK in its native chromosomal locus under anhydrotetracycline-dependent transcriptional control to establish the correlation between HPrK/P amounts and the efficiency of CCR in Bacillus subtilis. This resulted in about eightfold repression of HPrK/P expression but had no effect on CCR as monitored by xynP'-lacZ reporter gene expression and by analysis of RocG protein amounts. These results suggest that very small amounts of HPrK/P are sufficient for complete CCR and that control of HPrK/P activity depends only on the presence of effectors and not on the abundance of the enzyme.