Visualization of tandem repeat mutagenesis in Bacillus subtilis.

Mutations are crucial for the emergence and evolution of proteins with novel functions, and thus for the diversity of life. Tandem repeats (TRs) are mutational hot spots that are present in the genomes of all organisms. Understanding the molecular mechanism underlying TR mutagenesis at the level of single cells requires the development of mutation reporter systems. Here, we present a mutation reporter system that is suitable to visualize mutagenesis of TRs occurring in single cells of the Gram-positive model bacterium Bacillus subtilis using microfluidic single-cell cultivation. The system allows measuring the elimination of TR units due to growth rate recovery. The cultivation of bacteria carrying the mutation reporter system in microfluidic chambers allowed us for the first time to visualize the emergence of a specific mutation at the level of single cells. The application of the mutation reporter system in combination with microfluidics might be helpful to elucidate the molecular mechanism underlying TR (in)stability in bacteria. Moreover, the mutation reporter system might be useful to assess whether mutations occur in response to nutrient starvation.

Evidence for synergistic control of glutamate biosynthesis by glutamate dehydrogenases and glutamate in Bacillus subtilis.

In the Gram-positive bacterium, Bacillus subtilis glutamate is synthesized by the glutamine synthetase and the glutamate synthase (GOGAT). During growth with carbon sources that exert carbon catabolite repression, the rocG glutamate dehydrogenase (GDH) gene is repressed and the transcription factor GltC activates the expression of the GOGAT encoding gltAB genes. In the presence of amino acids of the glutamate family, the GDH RocG is synthesized and the enzyme prevents GltC from binding to DNA. The dual control of glutamate biosynthesis allows the efficient utilization of the available nutrients. Here we provide genetic and biochemical evidence that, like RocG, also the paralogous GDH GudB can inhibit the transcription factor GltC, thereby controlling glutamate biosynthesis. Contradictory previous observations show that high level of GDH activity does not result in permanent inhibition of GltC. By controlling the intracellular levels of glutamate through feeding with exogenous arginine, we observed that the GDH-dependent control of GltC and thus expression of the gltAB genes inversely correlates with the glutamate pool. These results suggest that the B. subtilis GDHs RocG and GudB in fact act as glutamate sensors. In conclusion, the GDH-mediated control of glutamate biosynthesis seems to depend on the intracellular glutamate concentration.

A novel engineering tool in the Bacillus subtilis toolbox: inducer-free activation of gene expression by selection-driven promoter decryptification.

Bacillus subtilis is a Gram-positive bacterium that is easy to manipulate genetically. Several methods for genome engineering have been developed that helped to extend our understanding of how the B. subtilis cell operates. Consequently, the bacterium has become one of the best-studied organisms. B. subtilis has also been engineered for industrial applications. Moreover, great progress has been achieved in promoter engineering to improve the performance of production strains. To expand the toolbox for engineering B. subtilis, we have constructed a system for the inducer-free activation of gene expression. The system relies on spontaneous mutational activation of a cryptic promoter and selection-driven enrichment of bacteria harbouring the mutated promoter. The synthetic promoter is cryptic due to a perfect direct repeat, separating the binding motifs of the RNA polymerase housekeeping sigma factor. The promoter can be fused to genes for industrial applications and to a growth-promoting gene that, upon mutational activation of the promoter, allows enrichment of the engineered bacteria due to a selective growth advantage.

Monitoring intraspecies competition in a bacterial cell population by cocultivation of fluorescently labelled strains.

Many microorganisms such as bacteria proliferate extremely fast and the populations may reach high cell densities. Small fractions of cells in a population always have accumulated mutations that are either detrimental or beneficial for the cell. If the fitness effect of a mutation provides the subpopulation with a strong selective growth advantage, the individuals of this subpopulation may rapidly outcompete and even completely eliminate their immediate fellows. Thus, small genetic changes and selection-driven accumulation of cells that have acquired beneficial mutations may lead to a complete shift of the genotype of a cell population. Here we present a procedure to monitor the rapid clonal expansion and elimination of beneficial and detrimental mutations, respectively, in a bacterial cell population over time by cocultivation of fluorescently labeled individuals of the Gram-positive model bacterium Bacillus subtilis. The method is easy to perform and very illustrative to display intraspecies competition among the individuals in a bacterial cell population.

The γ-aminobutyrate permease GabP serves as the third proline transporter of Bacillus subtilis.

PutP and OpuE serve as proline transporters when this imino acid is used by Bacillus subtilis as a nutrient or as an osmostress protectant, respectively. The simultaneous inactivation of the PutP and OpuE systems still allows the utilization of proline as a nutrient. This growth phenotype pointed to the presence of a third proline transport system in B. subtilis. We took advantage of the sensitivity of a putP opuE double mutant to the toxic proline analog 3,4-dehydro-dl-proline (DHP) to identify this additional proline uptake system. DHP-resistant mutants were selected and found to be defective in the use of proline as a nutrient. Whole-genome resequencing of one of these strains provided the lead that the inactivation of the γ-aminobutyrate (GABA) transporter GabP was responsible for these phenotypes. DNA sequencing of the gabP gene in 14 additionally analyzed DHP-resistant strains confirmed this finding. Consistently, each of the DHP-resistant mutants was defective not only in the use of proline as a nutrient but also in the use of GABA as a nitrogen source. The same phenotype resulted from the targeted deletion of the gabP gene in a putP opuE mutant strain. Hence, the GabP carrier not only serves as an uptake system for GABA but also functions as the third proline transporter of B. subtilis. Uptake studies with radiolabeled GABA and proline confirmed this conclusion and provided information on the kinetic parameters of the GabP carrier for both of these substrates.

Factors that mediate and prevent degradation of the inactive and unstable GudB protein in Bacillus subtilis.

The Gram-positive model bacterium Bacillus subtilis contains two glutamate dehydro genase-encoding genes, rocG and gudB. While the rocG gene encodes the functional GDH, the gudB gene is cryptic (gudB(CR) ) in the laboratory strain 168 due to a perfect 18 bp-long direct repeat that renders the GudB enzyme inactive and unstable. Although constitutively expressed the GudB(CR) protein can hardly be detected in B. subtilis as it is rapidly degraded within stationary growth phase. Its high instability qualifies GudB(CR) as a model substrate for studying protein turnover in B. subtilis. Recently, we have developed a visual screen to monitor the GudB(CR) stability in the cell using a GFP-GudB(CR) fusion. Using fluorescent microscopy we found that the GFP protein is simultaneously degraded together with GudB(CR). This allows us to analyze the stability of GudB(CR) in living cells. By combining the visual screen with a transposon mutagenesis approach we looked for mutants that show an increased fluorescence signal compared to the wild type indicating a stabilized GFP-GudB(CR) fusion. We observed, that disruption of the arginine kinase encoding gene mcsB upon transposon insertion leads to increased amounts of the GFP-GudB(CR) fusion in this mutant. Deletion of the cognate arginine phosphatase YwlE in contrast results in reduced levels of the GFP-GudB(CR) fusion. Recently, it was shown that the kinase McsB is involved in phosphorylation of GudB(CR) on arginine residues. Here we show that selected arginine-lysine point mutations of GudB(CR) exhibit no influence on degradation. The activity of McsB and YwlE, however, are crucial for the activation and inhibition, respectively, of a proteolytic machinery that efficiently degrades the unstable GudB(CR) protein in B. subtilis.

Selection-driven accumulation of suppressor mutants in bacillus subtilis: the apparent high mutation frequency of the cryptic gudB gene and the rapid clonal expansion of gudB(+) suppressors are due to growth under selection.

Soil bacteria like Bacillus subtilis can cope with many growth conditions by adjusting gene expression and metabolic pathways. Alternatively, bacteria can spontaneously accumulate beneficial mutations or shape their genomes in response to stress. Recently, it has been observed that a B. subtilis mutant lacking the catabolically active glutamate dehydrogenase (GDH), RocG, mutates the cryptic gudB(CR) gene at a high frequency. The suppressor mutants express the active GDH GudB, which can fully replace the function of RocG. Interestingly, the cryptic gudB(CR) allele is stably inherited as long as the bacteria synthesize the functional GDH RocG. Competition experiments revealed that the presence of the cryptic gudB(CR) allele provides the bacteria with a selective growth advantage when glutamate is scarce. Moreover, the lack of exogenous glutamate is the driving force for the selection of mutants that have inactivated the active gudB gene. In contrast, two functional GDHs are beneficial for the cells when glutamate was available. Thus, the amount of GDH activity strongly affects fitness of the bacteria depending on the availability of exogenous glutamate. At a first glance the high mutation frequency of the cryptic gudB(CR) allele might be attributed to stress-induced adaptive mutagenesis. However, other loci on the chromosome that could be potentially mutated during growth under the selective pressure that is exerted on a GDH-deficient mutant remained unaffected. Moreover, we show that a GDH-proficient B. subtilis strain has a strong selective growth advantage in a glutamate-dependent manner. Thus, the emergence and rapid clonal expansion of the active gudB allele can be in fact explained by spontaneous mutation and growth under selection without an increase of the mutation rate. Moreover, this study shows that the selective pressure that is exerted on a maladapted bacterium strongly affects the apparent mutation frequency of mutational hot spots.

Molecular diversity of phototrophic biofilms on building stone.

Composition and diversity of aeroterrestrial phototrophic microbial communities are up to now poorly understood. Here, we present a comparative study addressing the composition of algal communities on sandstone substrata based upon the analysis of rRNA gene clone libraries from environmental samples and crude cultures. From a west-facing, shaded wall area of the mediaeval castle ruin Gleichen (Thuringia, Germany), sequences mainly related to the green algae Prasiococcus and Trebouxia (Trebouxiophyceae) were retrieved. A south-west-facing, sun-exposed wall area was mainly colonized by Apatococcus and a Phyllosiphon-related alga. Just a few species, in particular Stichococcus-related strains, were ubiquitous in both areas. Samples from a basement vault exposed to low irradiance exhibited Chlorophyceae like Chromochloris and Bracteacoccus. Thus, most green algae on the daylight-exposed walls were affiliated to Trebouxiophyceae, whereas Chlorophyceae were dominant in samples taken from the site kept under low irradiance. Accordingly, cyanobacterial communities were different: the sun-exposed area was dominated by Synechococcus-related organisms, while on the shaded wall area, cyanobacteria were almost absent. The filamentous Leptolyngbya dominated samples from the basement vault. Scanning electron microscopy revealed endolithic algal morphotypes (coccoid algae and diatoms) dominant in open pores between mineral particles. Here, the organisms may be also involved in biogenic weathering of stone.

The fingerprint of chemosymbiosis: origin and preservation of isotopic biosignatures in the nonseep bivalve Loripes lacteus compared with Venerupis aurea.

Endosymbionts in marine bivalves leave characteristic biosignatures in their host organisms. Two nonseep bivalve species collected in Mediterranean lagoons, thiotrophic symbiotic Loripes lacteus and filter-feeding nonsymbiotic Venerupis aurea, were studied in detail with respect to generation and presence of such signatures in living animals, and the preservation of these signals in subfossil (late Pleistocene) sedimentary shells. Three key enzymes from sulfur oxidation (APS-reductase), CO(2) fixation (RubisCO) and assimilation of nitrogen [glutamine synthetase (GS)] were detected by immunofluorescence in the bacterial symbionts of Loripes. In Loripes, major activity was derived from GS of the symbionts whereas in Venerupis the host GS is active. In search of geologically stable biosignatures for thiotrophic chemosymbiosis that might be suitable to detect such associations in ancient bivalves, we analyzed the isotopic composition of shell lipids (δ(13)C) and the bulk organic matrix of the shell (δ(13)C , δ(15)N , δ(34)S). In the thiotrophic Loripes, δ(13)C values were depleted compared with the filter-feeding Venerupis by as much as 8.5‰ for individual fatty acids, and 4.4‰ for bulk organic carbon. Likewise, bulk δ(15)N and δ(34)S values were more depleted in recent thiotrophic Loripes. Whereas δ (34)S values were found to be unstable over time, the combined δ(15)N and δ(13)C values in organic shell extracts revealed a specific signature for chemosymbiosis in recent and subfossil specimens.