Insights into the Roles of Lipoteichoic Acids and MprF in Bacillus subtilis.

Gram-positive bacterial cells are protected from the environment by a cell envelope that is comprised of a thick layer of peptidoglycan that maintains cell shape and teichoic acid polymers whose biological function remains unclear. In Bacillus subtilis, the loss of all class A penicillin-binding proteins (aPBPs), which function in peptidoglycan synthesis, is conditionally lethal. Here, we show that this lethality is associated with an alteration of lipoteichoic acids (LTAs) and the accumulation of the major autolysin LytE in the cell wall. Our analysis provides further evidence that the length and abundance of LTAs act to regulate the cellular level and activity of autolytic enzymes, specifically LytE. Importantly, we identify a novel function for the aminoacyl-phosphatidylglycerol synthase MprF in the modulation of LTA biosynthesis in both B. subtilis and Staphylococcus aureus. This finding has implications for our understanding of antimicrobial resistance (particularly to daptomycin) in clinically relevant bacteria and the involvement of MprF in the virulence of pathogens such as methicillin-resistant S. aureus (MRSA). IMPORTANCE In Gram-positive bacteria such as Bacillus subtilis and Staphylococcus aureus, the cell envelope is a structure that protects the cells from the environment but is also dynamic in that it must be modified in a controlled way to allow cell growth. In this study, we show that lipoteichoic acids (LTAs), which are anionic polymers attached to the membrane, have a direct role in modulating the cellular abundance of cell wall-degrading enzymes. We also find that the apparent length of the LTA is modulated by the activity of the enzyme MprF, previously implicated in modifications of the cell membrane leading to resistance to antimicrobial peptides. These findings are important contributions to our understanding of how bacteria balance cell wall synthesis and degradation to permit controlled growth and division. These results also have implications for the interpretation of antibiotic resistance, particularly for the clinical treatment of MRSA infections.

Mild hydrostatic pressure triggers oxidative responses in Escherichia coli.

Hydrostatic pressure is an important physical stimulus which can cause various responses in bacterial cells. The survival and cellular processes of Escherichia coli under hydrostatic pressures between 10 MPa and 110 MPa have been studied. However, understanding bacterial responses to moderately elevated pressure of up to 10 MPa is useful for a range of different applications including for example in smart and responsive materials. In this study, the genetic responses of E. coli K-12 MG1655 to 1 MPa pressure was examined using transcriptomic analysis by RNA-Seq. The results show that 101 genes were differentially expressed under 1 MPa pressure in E. coli cells, with 85 of them up-regulated. The analysis suggested that some genes were over expressed to adapt the increase of oxygen levels in our system, and several functional categories are involved including oxidative stress responses, Fe-S cluster assembly and iron acquisition. Two differentially expressed genes azuC and entC were further investigated using RT-qPCR, and GFP reported strains of those two genes were created, AG1319 (PazuC azuC-msfgfp) and AG1321 (PentC entC-msfgfp). A linear response of azuC expression was observed between 0 MPa to 1 MPa by monitoring the fluorescence signal of strain AG1319 (PazuC azuC-msfgfp). This study is the first report to demonstrate the genetic response of bacterial cells under 1 MPa hydrostatic pressure, and provides preliminary data for creating pressure sensing bacterial strains for a wide range of applications.

Design and modelling of an engineered bacteria-based, pressure-sensitive soil.

In this paper, we describe the first steps in the design of a synthetic biological system based on the use of genetically modified bacteria to detect elevated pressures in soils and respond by cementing soil particles. Such a system might, for example, enable a self- constructed foundation to form in response to load using engineered bacteria which could be seeded and grown in the soils. This process would reduce the need for large-scale excavations and may be the basis for a new generation of self-assembling and responsive bio-based materials. A prototype computational model is presented which integrates experimental data from a pressure sensitive gene within Escherichia coli bacteria with geotechnical models of soil loading and pore water pressure. The results from the integrated model are visualised by mapping expected gene expression values onto the soil volume. We also use our experimental data to design a two component system where one type of bacteria acts as a sensor and signals to another material synthesis bacteria. The simulation demonstrates the potential of computational models which integrate multiple scales from macro stresses in soils to the expression of individual genes to inform new types of design process. The work also illustrates the combination of in silico (silicon based computing) computation with in vivo (in the living) computation.

RodA as the missing glycosyltransferase in Bacillus subtilis and antibiotic discovery for the peptidoglycan polymerase pathway.

The bacterial cell wall is a highly conserved essential component of most bacterial groups. It is the target for our most frequently used antibiotics and provides important small molecules that trigger powerful innate immune responses. The wall is composed of glycan strands crosslinked by short peptides. For many years, the penicillin-binding proteins were thought to be the key enzymes required for wall synthesis. RodA and possibly other proteins in the wider SEDS (shape, elongation, division and sporulation) family have now emerged as a previously unknown class of essential glycosyltranferase enzymes, which play key morphogenetic roles in bacterial cell wall synthesis. We provide evidence in support of this role and the discovery of small natural product molecules that probably target these enzymes. The SEDS proteins have exceptional potential as targets for new antibacterial therapeutic agents.

Identified members of the Streptomyces lividans AdpA regulon involved in differentiation and secondary metabolism.

BACKGROUND: AdpA is a key transcriptional regulator involved in the complex growth cycle of Streptomyces. Streptomyces are Gram-positive bacteria well-known for their production of secondary metabolites and antibiotics. Most work on AdpA has been in S. griseus, and little is known about the pathways it controls in other Streptomyces spp. We recently discovered interplay between ClpP peptidases and AdpA in S. lividans. Here, we report the identification of genes directly regulated by AdpA in S. lividans. RESULTS: Microarray experiments revealed that the expression of hundreds of genes was affected in a S. lividans adpA mutant during early stationary phase cultures in YEME liquid medium. We studied the expression of the S. lividans AdpA-regulated genes by quantitative real-time PCR analysis after various times of growth. In silico analysis revealed the presence of potential AdpA-binding sites upstream from these genes; electrophoretic mobility shift assays indicated that AdpA binds directly to their promoter regions. This work identifies new pathways directly controlled by AdpA and that are involved in S. lividans development (ramR, SLI7885 also known as hyaS and SLI6586), and primary (SLI0755-SLI0754 encoding CYP105D5 and Fdx4) or secondary (cchA, cchB, and hyaS) metabolism. CONCLUSIONS: We characterised six S. lividans AdpA-dependent genes whose expression is directly activated by this pleiotropic regulator. Several of these genes are orthologous to bldA-dependent genes in S. coelicolor. Furthermore, in silico analysis suggests that over hundred genes may be directly activated or repressed by S. lividans AdpA, although few have been described as being part of any Streptomyces AdpA regulons. This study increases the number of known AdpA-regulated pathways in Streptomyces spp.

Regulation of the clpP1clpP2 operon by the pleiotropic regulator AdpA in Streptomyces lividans.

Insertion of an apramycin resistance cassette in the clpP1clpP2 operon (encoding the ClpP1 and ClpP2 peptidase subunits) affects morphological and physiological differentiation of Streptomyces lividans. Another key factor controlling Streptomyces differentiation is the pleiotropic transcriptional regulator AdpA. We have identified a spontaneous missense mutation (-1 frameshift) in the adpA (bldH) open reading frame in a clpP1clpP2 mutant that led to the synthesis of a non-functional AdpA protein. Electrophoretic mobility shift assays showed that AdpA bound directly to clpP1clpP2 promoter region. Quantitative real-time PCR analysis showed that AdpA regulated the clpP1clpP2 operon expression at specific growth times. In vitro, AdpA and ClgR, a transcriptional activator of clpP1clpP2 operon and other genes, were able to bind simultaneously to clpP1 promoter, which suggests that AdpA binding to clpP1 promoter did not affect that of ClgR. This study allowed to uncover an interplay between the ClpP peptidases and AdpA in S. lividans.