Transcriptional Profile of Bacillus subtilis sigF-Mutant during Vegetative Growth

Sigma factor F is the first forespore specific transcription factor in Bacillus subtilis and controls genes required for the early stages of prespore development. The role of sigF is well studied under conditions that induce sporulation. Here, the impact of sigF disruption on the transcriptome of exponentially growing cultures is studied by micro-array analysis. Under these conditions that typically don't induce sporulation, the transcriptome showed minor signs of sporulation initiation. The number of genes differentially expressed and the magnitude of expression were, as expected, quite small in comparison with sporulation conditions. The genes mildly down-regulated were mostly involved in anabolism and the genes mildly up-regulated, in particular fatty acid degradation genes, were mostly involved in catabolism. This is probably related to the arrest at sporulation stage II occurring in the sigF mutant, because continuation of growth from the formed disporic sporangia may require additional energy. The obtained knowledge is relevant for various experiments, such as industrial fermentation, prolonged experimental evolution or zero-growth studies, where sporulation is an undesirable trait that should be avoided, e.g by a sigF mutation

Bacillus subtilis at near-zero specific growth rates: adaptations to extreme caloric restriction

Bacillus subtilis is an important soil-dwelling bacteria species that is used for the production of e.g. vitamins, enzymes and medicines. In both the natural and industrial environment the availability of energy sources can be limited. In contrary to a situation of complete ‘nutrient depletion’, many microbial communities live under conditions of ‘hunger’, because the concentration of available energy sources usually is not completely zero. Consequently, in these circumstances microorganisms grow with an extremely low growth rate, especially in comparison with laboratory cultivation. To better understand microbial life, the study of adaption to such conditions is necessary. In this thesis the study is described of B. subtilis at near-zero growth rates. These extremely low growth rates are achieved by extreme caloric restriction in a so-called retentostat system. Despite these harsh conditions nearly all bacteria remained viable. Calculations showed that almost all the available energy was used for cellular processes that were related to maintenance of the cell, and not for growth. Analysis of gene expression levels revealed that cells reprogram their gene expression to increase their chance of survival. This response to ‘hunger’ has similarities with the response to complete nutrient depletion, but differs in some areas. Additionally, this thesis describes i.e. the study and upgrade of the Green Fluorescent Protein ‘toolbox’ for B. subtilis, Lactococcus lactis and Streptococcus pneumoniae. Green Fluorescent Protein (GFP) is an important tool in molecular biology to visualize gene expression and protein localisation

Bacterial strains and plasmids used in this study^a.

<p>^a Abbreviations: Amp^r, ampicillin resistance; Sp^r, spectinomycin resistance</p><p>Bacterial strains and plasmids used in this study<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141553#t001fn001" target="_blank">^a</a>.</p

FIVA analysis of <i>B</i>. <i>subtilis</i> 168 <i>sigF</i>::<i>spc</i> gene expression under vegetative conditions.

<p>Genes from one DNA microarray dataset (<i>sigF</i> mutant strain compared to wild-type strain during exponential growth) were partitioned into up- and down-regulated clusters. The size of each cluster is displayed in blue underneath the cluster name. Numbers in each rectangle represent absolute values of occurrences. The significance of occurrences is visualized in a colour gradient that is displayed at the bottom of the plot. The description of each category is placed at the right. Multiple testing correction results are visualized using five different symbols to distinguish between the individual corrections. The number of symbols placed in each rectangle corresponds to the number of multiple testing corrections after which the annotation is found significant. This figure legend is cited from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141553#pone.0141553.ref033" target="_blank">33</a>].</p

Oligonucleotides used in this study.

<p>Restriction sites are underlined.</p><p>Oligonucleotides used in this study.</p

Influence of Shifting Positions of Ser, Thr, and Cys Residues in Prenisin on the Efficiency of Modification Reactions and on the Antimicrobial Activities of the Modified Prepeptides▿ †

Since the recent discovery that the nisin modification and transport machinery can be used to produce and modify peptides unrelated to nisin, specific questions arose concerning the specificity of the modification enzymes involved and the limits of their promiscuity with respect to the dehydration and cyclization processes. The nisin leader peptide has been postulated to fulfill a recognition and binding function required for these modifications. Here, we investigated whether the relative positions of the modifiable residues in the nisin prepeptide, with respect to the leader peptide, could influence the efficiency of their modification. We conducted a systematic study on the insertion of one to four alanines in front of either ring A or ring D to change the “reading frame” of modifiable residues, resulting in altered distance and topology of the modifiable residues relative to the leader. The insertion of N-terminal and hinge-located Ala residues had only a modest influence on the modification efficiency, demonstrating that the “phasing” of these residues relative to the leader peptide is not a critical factor in determining modification. However, in all cases, but especially with the N-terminal insertions, the antimicrobial activities of the fully modified nisin species were decreased

Physiological and cell morphology adaptation of Bacillus subtilis at near-zero specific growth rates: a transcriptome analysis

Nutrient scarcity is a common condition in nature, but the resulting extremely low growth rates (below 0.025 h(-1) ) are an unexplored research area in Bacillus subtilis. To understand microbial life in natural environments, studying the adaptation of B. subtilis to near-zero growth conditions is relevant. To this end, a chemostat modified for culturing an asporogenous B. subtilis sigF mutant strain at extremely low growth rates (also named a retentostat) was set up, and biomass accumulation, culture viability, metabolite production and cell morphology were analysed. During retentostat culturing, the specific growth rate decreased to a minimum of 0.00006 h(-1) , corresponding to a doubling time of 470 days. The energy distribution between growth and maintenance-related processes showed that a state of near-zero growth was reached. Remarkably, a filamentous cell morphology emerged, suggesting that cell separation is impaired under near-zero growth conditions. To evaluate the corresponding molecular adaptations to extremely low specific growth, transcriptome changes were analysed. These revealed that cellular responses to near-zero growth conditions share several similarities with those of cells during the stationary phase of batch growth. However, fundamental differences between these two non-growing states are apparent by their high viability and absence of stationary phase mutagenesis under near-zero growth conditions