OT-Mation is an open-source Python script designed to automate the programming of OT-2 liquid-handling robots, making combinatorial experiments more accessible to researchers. By parsing user-defined CSV files containing information on labware, reagents, pipettes, and experimental design, OT-Mation generates a bespoke Python script compatible with the OT-2 system. OT-Mation enhances reproducibility, reduces human error, and streamlines workflows, making it a valuable addition to any laboratory utilizing OT-2 robotics for liquid handling. While OT-Mation can be used for setting up any type of experiment on the OT-2, its real utility lies in making the connection between multifactorial experimental design software outputs (i.e. design of experiments arrays) and liquid-handling robot executable code. As such, OT-Mation helps bridge the gap between code-based flexibility and user-friendly operation, allowing researchers with limited programming skills to design and execute complex experiments efficiently. Graphical Abstract.

Plant-associated endophytes offer promising agricultural, environmental, and biotechnological applications. Despite their potential utility, difficulties in culturing these microorganisms under laboratory conditions have limited both their isolation and a comprehensive understanding of their biology, function, and ecological role. Against this background, Pseudomonas granadensis strain CT364—isolated from the olive tree rhizosphere—emerged as a potential endophyte of interest due to its cultivability and its ability to promote rooting across diverse species, including olive trees, rapeseed, mung bean and cowpea. Genome Annotation and in silico predictions identified 564 genes linked to rhizosphere competence, plant colonisation and plant growth-promoting traits. Experimental findings confirmed the strain’s motility, capacity for biofilm formation, and ability to sense and respond to plant-derived signals. P. granadensis CT364 effectively colonises the rhizosphere, rhizoplane, and internal tissues of Arabidopsis, confirming its endophytic nature without exhibiting any pathogenic traits. Inoculation experiments demonstrated significant effects on root architecture and increases in plant biomass and rosette area. Notably, these benefits were retained under salinity and osmotic stress, underscoring its plant growth-promoting ability. Finally, both genome analysis and experimental tests confirmed its resistance to osmotic stress and heavy metal toxicity, highlighting the strain’s ability to survive in difficult environments. The integration of genomic insights and experimental validation supports the conclusion that P. granadensis CT364 is a plant growth-promoting endophytic bacterium. Its ability to enhance plant development under both optimal and stressful conditions, combined with its ability to colonise Arabidopsis and non-pathogenic nature, positions this strain as a potential bioinoculant for sustainable agriculture. Furthermore, the identification of specific genes related to plant sensing and colonisation, and its genetic tractability, open avenues for exploring underlying mechanisms of plant–microbe interactions. In summary, P. granadensis CT364 therefore not only holds potential for improving crop performance under challenging environmental conditions but also offers a valuable model for the study of beneficial plant–bacterial symbiosis.

Pseudomonas sp. CT364 was isolated from olive tree rhizosphere in Seville (Spain). We report its complete genome sequence, acquired by co-assembling Illumina and Nanopore reads. The genome comprises a circular chromosome of 6.2 Mbp and a G + C content of 60.0%. Taxonomic analyses confirmed it to be Pseudomonas granadensis.

The importance of genomics in the COVID-19 age cannot be overstated and genomics has a key place in the advanced undergraduate microbiology curriculum. The COVID-19 pandemic, and its attendant lockdowns, have necessitated a change in the delivery of our microbial genomics module. The key challenges for delivering a remote computer-based genomics module, are ensuring student engagement and learning; and supporting the technical aspects of remote computer work. In the absence of in-person classes, we have adapted our material covering the principles and theory of microbial genomics, to asynchronous videos and synchronous online workshops. Practical training for all undergraduate microbiologists in the UK has been severely reduced, which has forced us to focus skills training towards computational aspects of genome assembly, analysis, and interpretation. The set up and implementation of a robust and scalable Linux-based genome analysis pipeline for students presents many challenges: from organising remote access to computer clusters; software support; and managing hardware. Assessment for the module is based on the demonstrating learning and skills development through the analysis of SARS-CoV2 genomes to identify spike protein variation and mapping this to published structures. Terminal assessment is through the analysis of newly sequenced bacterial genomes and the preparation of a genome report suitable for publication. We will outline the implementation and management of our Microbial Genomics module as a model for computer-based skills training for undergraduate microbiologists. Furthermore, we will discuss the impact of the COVID-19 pandemic on the module and the opportunities it has presented.

Abyssomicin C is a polyketide antibiotic produced by Micromonospora maris AB-18-032. Previous work on abyssomicin C indicated that it inhibits growth of infectious pathogens such as Methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA). It does this by suppressing para-aminobenzoic acid (pABA) synthesis, which is required for folic acid biosynthesis in bacteria. This makes abyssomicin C an appealing antibiotic drug, as it is specific only to bacteria. However, its yield in chemical synthesis (4 %) and biosynthesis (60.0 mg/L) are low. Ribosome engineering, through the selection of streptomycin-resistant and rifampin-resistant mutants, of M. maris may result in strains with a higher titer of production. After screening by bioassay and sequencing, the mutant genes in six Ochi mutants were identified. Of these mutants, four of them are able to produce a higher titer of abyssomicin C. The other two strains were detected to produce the other secondary metabolite.

EDITORIAL article Front. Microbiol., 01 May 2018 | https://doi.org/10.3389/fmicb.2018.00824

To combat infection and antimicrobial resistance, it is helpful to elucidate drug mechanism(s) of action. Here we examined how the widely used antimicrobial polyhexamethylene biguanide (PHMB) kills bacteria selectively over host cells. Contrary to the accepted model of microbial membrane disruption by PHMB, we observed cell entry into a range of bacterial species, and treated bacteria displayed cell division arrest and chromosome condensation, suggesting DNA binding as an alternative antimicrobial mechanism. A DNA-level mechanism was confirmed by observations that PHMB formed nanoparticles when mixed with isolated bacterial chromosomal DNA and its effects on growth were suppressed by pairwise combination with the DNA binding ligand Hoechst 33258. PHMB also entered mammalian cells, but was trapped within endosomes and excluded from nuclei. Therefore, PHMB displays differential access to bacterial and mammalian cellular DNA and selectively binds and condenses bacterial chromosomes. Because acquired resistance to PHMB has not been reported, selective chromosome condensation provides an unanticipated paradigm for antimicrobial action that may not succumb to resistance.

Peptide nucleic acids (PNAs) are a class of artificial DNA/RNA analogues that have unique physicochemical properties, which include a high chemical stability, resistance to nucleases and proteases and higher mismatch sensitivity than DNA. PNAs were initially anticipated to be useful for application in antisense and antigene therapies; however, their poor cellular uptake has limited their use for such purposes in the "real world". Recently, it has been shown that the addition of metal complexes to these oligonucleotide analogues could open up new avenues for their utilization in various research fields. Such metallo-constructs have shown great promise, for a diverse range of applications, most notably in the biosensing area. In this chapter, we report on the recent synthetic advances towards the preparation of these "(multi)-metallic PNAs" on the solid phase.

This chapter reviews current knowledge on the structure, biochemistry and function of the FtsZ protein in bacterial pathogens, highlighting its potential as a promising drug target for the development of novel antibacterial agents. Strategies that can be utilized for the identification of potential Ftsz inhibitors are discussed.

Background Berberine is a plant alkaloid that is widely used as an anti-infective in traditional medicine. Escherichia coli exposed to berberine form filaments, suggesting an antibacterial mechanism that involves inhibition of cell division. Berberine is a DNA ligand and may induce filamentation through induction of the SOS response. Also, there is biochemical evidence for berberine inhibition of the cell division protein FtsZ. Here we aimed to assess possible berberine mechanism(s) of action in growing bacteria using genetics tools. Methodology/Principal Findings First, we tested whether berberine inhibits bacterial growth through DNA damage and induction of the SOS response. The SOS response induced by berberine was much lower compared to that induced by mitomycin C in an SOS response reporter strain. Also, cell filamentation was observed in an SOS-negative E. coli strain. To test whether berberine inhibits FtsZ, we assessed its effects on formation of the cell division Z-rings, and observed a dramatic reduction in Z-rings in the presence of berberine. We next used two different strategies for RNA silencing of ftsZ and both resulted in sensitisation of bacteria to berberine, visible as a drop in the Minimum Inhibitory Concentration (MIC). Furthermore, Fractional Inhibitory Concentration Indices (FICIs) showed a high level of synergy between ftsZ silencing and berberine treatment (FICI values of 0.23 and 0.25 for peptide nucleic acid- and expressed antisense RNA-based silencing of ftsZ, respectively). Finally, over-expression of ftsZ led to a mild rescue effect in berberine-treated cells. Conclusions The results argue against DNA binding as the primary mechanism of action of berberine and support the hypothesis that its antibacterial properties are due to inhibition of the cell division protein FtsZ. In addition, the genetic approach used here provides a means to rapidly test the activity of other putative FtsZ inhibitors.

Background: Genes essential for bacterial growth are of particular scientific interest. Many putative essential genes have been identified or predicted in several species, however, little is known about gene expression requirement stringency, which may be an important aspect of bacterial physiology and likely a determining factor in drug target development.Methodology/Principal Findings: Working from the premise that essential genes differ in absolute requirement for growth, we describe silencing of putative essential genes in E. coli to obtain a titration of declining growth rates and transcript levels by using antisense peptide nucleic acids (PNA) and expressed antisense RNA. The relationship between mRNA decline and growth rate decline reflects the degree of essentiality, or stringency, of an essential gene, which is here defined by the minimum transcript level for a 50% reduction in growth rate (MTL50). When applied to four growth essential genes, both RNA silencing methods resulted in MTL50 values that reveal acpP as the most stringently required of the four genes examined, with ftsZ the next most stringently required. The established antibacterial targets murA and fabI were less stringently required.Conclusions: RNA silencing can reveal stringent requirements for gene expression with respect to growth. This method may be used to validate existing essential genes and to quantify drug target requirement.

This study compared different methods of direct DNA extraction and purification from a silt loam soil and investigated the relationship between DNA quantity and sequence diversity. Five extraction methods and four purification techniques were investigated. Quantities of DNA extracted were between 3.4+/-0.55 and 54.3+/-8.18 &mgr;g g(-1) (dry wt) of soil with OD(260)/OD(230) purity ratios between 0.80 and 1.15. Analysis of sequence diversity in all extracts was conducted using PCR-single strand conformation polymorphism (SSCP). Profiles generated using universal 16S rDNA primers (Com1/Com2) were found to be identical when used to amplify 16S rDNA extracted directly from soil. The genus Pseudomonas was targeted in order to reduce profile complexity, which was apparent when using universal 16S rDNA primers, and which hindered direct comparison of sequence diversity. A Pseudomonas culture library and non-cultured Pseudomonas 16S rDNA genes were used to provide a background count of Pseudomonas operational taxonomic units present in the soil. Cloning and sequencing of amplicons generated using a Pseudomonas-specific (Ps-for) and a universal 16S rDNA (Com2) primer, coupled with nested amplification (Com1/Com2 amplification from Ps-for/Ps-rev amplicons), used in conjunction with SSCP, revealed that environmental contaminants co-extracted with DNA, such as humic acid, significantly reduced primer specificity. SSCP was sensitive enough to reveal template bias in different primer sets. PCR-restriction fragment length-SSCP of Pseudomonas 16S rDNA amplified from soil-extracted DNA revealed distinct differences in sequence representation between extraction methods and showed that greater DNA yield is not synonymous with higher sequence diversity. We, therefore, suggest that DNA extractions from soil should be evaluated not only in terms of quantity and purity, but also in terms of the sequence diversity present. SSCP proved to be a valuable tool for the assessment of the methodologies commonly used in PCR-mediated microbial ecology studies.