Bacillus cytotoxicus Genomics: Chromosomal Diversity and Plasmidome Versatility

Bacillus cytotoxicus is the thermotolerant representative of the Bacillus cereus group. This group, also known as B. cereus sensu lato, comprises both beneficial and pathogenic members and includes psychrotolerant and thermotolerant species. Bacillus cytotoxicus was originally recovered from a fatal outbreak in France in 1998. This species forms a remote cluster from the B. cereus group members and reliably contains the cytk-1 gene, coding for a cytotoxic variant of cytotoxin K. Although this species was originally thought to be homogenous, intra-species diversity has been recently described with four clades, six random amplified polymorphic DNA (RAPD) patterns, and 11 plasmids profiles. This study aimed to get new insights into the genomic diversity of B. cytotoxicus and to decipher the underlying chromosomal and plasmidial variations among six representative isolates through whole genome sequencing (WGS). Among the six sequenced strains, four fitted the previously described genomic clades A and D, while the remaining two constituted new distinct branches. As for the plasmid content of these strains, three large plasmids were putatively conjugative and three small ones potentially mobilizable, harboring coding genes for putative leaderless bacteriocins. Mobile genetic elements, such as prophages, Insertion Sequences (IS), and Bacillus cereus repeats (bcr) greatly contributed to the B. cytotoxicus diversity. As for IS elements and bcr, IS3 and bcr1 were the most abundant elements and, along with the group II intron B.c.I8, were found in all analyzed B. cytotoxicus strains. When compared to other B. cytotoxicus strains, the type-strain NVH 391-98 displayed a relatively low number of IS. Our results shed new light on the contribution of mobile genetic elements to the genome plasticity of B. cytotoxicus and their potential role in horizontal gene transfer.

What makes a megaplasmid?

Naturally occurring plasmids come in different sizes. The smallest are less than a kilobase of DNA, while the largest can be over three orders of magnitude larger. Historically, research has tended to focus on smaller plasmids that are usually easier to isolate, manipulate and sequence, but with improved genome assemblies made possible by long-read sequencing, there is increased appreciation that very large plasmids—known as megaplasmids—are widespread, diverse, complex, and often encode key traits in the biology of their host microorganisms. Why are megaplasmids so big? What other features come with large plasmid size that could affect bacterial ecology and evolution? Are megaplasmids 'just' big plasmids, or do they have distinct characteristics? In this perspective, we reflect on the distribution, diversity, biology, and gene content of megaplasmids, providing an overview to these large, yet often overlooked, mobile genetic elements. This article is part of the theme issue ‘The secret lives of microbial mobile genetic elements’.

In vitro amplification of whole large plasmids via transposon-mediated oriC insertion

DNA amplification is a fundamental technique in molecular biology. The replication cycle reaction is a new method for amplification of large circular DNA having oriC sequences, which is a replication initiation site of the Escherichia coli chromosome. We here developed a replication cycle reaction-based method useful for amplification of various circular DNAs lacking oriC, even in the absence of any sequence information, via transposon-mediated oriC insertion to the circular DNA template. A 15-kb non- oriC plasmid was amplified from a very small amount of starting DNA (50 fg, 1 fM). The method was also applicable to GC-rich plasmid (69%) or large F-plasmid (230 kb). This method thus provides a powerful tool to amplify various environmental circular DNAs.

ORFograph: search for novel insecticidal protein genes in genomic and metagenomic assembly graphs

Background Since the prolonged use of insecticidal proteins has led to toxin resistance, it is important to search for novel insecticidal protein genes (IPGs) that are effective in controlling resistant insect populations. IPGs are usually encoded in the genomes of entomopathogenic bacteria, especially in large plasmids in strains of the ubiquitous soil bacteria, Bacillus thuringiensis (Bt). Since there are often multiple similar IPGs encoded by such plasmids, their assemblies are typically fragmented and many IPGs are scattered through multiple contigs. As a result, existing gene prediction tools (that analyze individual contigs) typically predict partial rather than complete IPGs, making it difficult to conduct downstream IPG engineering efforts in agricultural genomics. Methods Although it is difficult to assemble IPGs in a single contig, the structure of the genome assembly graph often provides clues on how to combine multiple contigs into segments encoding a single IPG. Results We describe ORFograph, a pipeline for predicting IPGs in assembly graphs, benchmark it on (meta)genomic datasets, and discover nearly a hundred novel IPGs. This work shows that graph-aware gene prediction tools enable the discovery of greater diversity of IPGs from (meta)genomes. Conclusions We demonstrated that analysis of the assembly graphs reveals novel candidate IPGs. ORFograph identified both already known genes “hidden” in assembly graphs and potential novel IPGs that evaded existing tools for IPG identification. As ORFograph is fast, one could imagine a pipeline that processes many (meta)genomic assembly graphs to identify even more novel IPGs for phenotypic testing than would previously be inaccessible by traditional gene-finding methods. While here we demonstrated the results of ORFograph only for IPGs, the proposed approach can be generalized to any class of genes.

The higBA- Type Toxin-Antitoxin System in IncC Plasmids Is a Mobilizable Ciprofloxacin-Inducible System

Toxin-antitoxin (TA) systems play vital roles in maintaining plasmids in bacteria. Plasmids with incompatibility group C are large plasmids that disseminate via conjugation and carry high-profile antibiotic resistance genes.

A high-efficiency method for site-directed mutagenesis of large plasmids based on large DNA fragment amplification and recombinational ligation

Site-directed mutagenesis for large plasmids is a difficult task that cannot easily be solved by the conventional methods used in many laboratories. In this study, we developed an effective method for Site-directed Mutagenesis for Large Plasmids (SMLP) based on a PCR technique. The SMLP method combines several effective approaches, including a high-efficiency DNA polymerase for the large DNA amplification, two independent PCR reactions and a fast recombinational ligation. Using this method, we have achieved a variety of mutants for the filamin A gene (7.9 kb) cloned in the pcDNA (5.4 kb) or the pLV-U6-CMV-EGFP (9.4 kb) plasmids, indicating that this method can be applied to site-directed mutagenesis for the plasmids up to 17.3 kb. We show that the SMLP method has a greater advantage than the conventional methods tested in this study, and this method can be applied to substitution, deletion, and insertion mutations for both large and small plasmids as well as the assembly of three fragments from PCR reactions. Altogether, the SMLP method is simple, effective, and beneficial to the laboratories that require completing the mutagenesis of large plasmids.

Plasmid fitness costs are caused by specific genetic conflicts

Plasmids play an important role in bacterial genome evolution by transferring genes between lineages. Fitness costs associated with plasmid acquisition are expected to be a barrier to gene exchange, but the causes of plasmid fitness costs are poorly understood. Single compensatory mutations are often sufficient to completely ameliorate plasmid fitness costs, suggesting that such costs are caused by specific genetic conflicts rather than generic properties of plasmids, such as their size, metabolic burden, or expression level. Here we show -- using a combination of experimental evolution, reverse genetics, and transcriptomics -- that fitness costs of two divergent large plasmids in Pseudomonas fluorescens are caused by inducing maladaptive expression of a chromosomal tailocin toxin operon. Mutations in single genes unrelated to the toxin operon, and located on either the chromosome or the plasmid, ameliorated the disruption associated with plasmid acquisition. We identify one of these compensatory loci, the chromosomal gene PFLU4242, as the key mediator of the fitness costs of both plasmids, with the other compensatory loci either reducing expression of this gene or mitigating its deleterious effects by upregulating a putative plasmid-borne ParAB operon. The chromosomal mobile genetic element Tn6291, which uses plasmids for transmission, remained upregulated even in compensated strains, suggesting that mobile genetic elements communicate through pathways independent of general physiological disruption. Plasmid fitness costs caused by specific genetic conflicts are unlikely to act as a long-term barrier to horizontal gene transfer due to their propensity for amelioration by single compensatory mutations, explaining why plasmids are so common in bacterial genomes.

Improved CRISPR/Cas9 Tools for the Rapid Metabolic Engineering of Clostridium acetobutylicum

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas (CRISPR-associated proteins)9 tools have revolutionized biology—several highly efficient tools have been constructed that have resulted in the ability to quickly engineer model bacteria, for example, Escherichia coli. However, the use of CRISPR/Cas9 tools has lagged behind in non-model bacteria, hampering engineering efforts. Here, we developed improved CRISPR/Cas9 tools to enable efficient rapid metabolic engineering of the industrially relevant bacterium Clostridium acetobutylicum. Previous efforts to implement a CRISPR/Cas9 system in C. acetobutylicum have been hampered by the lack of tightly controlled inducible systems along with large plasmids resulting in low transformation efficiencies. We successfully integrated the cas9 gene from Streptococcuspyogenes into the genome under control of the xylose inducible system from Clostridium difficile, which we then showed resulted in a tightly controlled system. We then optimized the length of the editing cassette, resulting in a small editing plasmid, which also contained the upp gene in order to rapidly lose the plasmid using the upp/5-fluorouracil counter-selection system. We used this system to perform individual and sequential deletions of ldhA and the ptb-buk operon.

Prevalence of Carbapenem-Resistant Klebsiella pneumoniae Co-Harboring blaKPC-Carrying Plasmid and pLVPK-Like Virulence Plasmid in Bloodstream Infections

This study aimed to characterize carbapenem-resistant Klebsiella pneumoniae (CR-KP) co-harboring blaKPC-2-carrying plasmid and pLVPK-like virulence plasmid. Between December 2017 and April 2018, 24 CR-KP isolates were recovered from 24 patients with bacteremia. The mortality was 66.7%. Pulsed-field gel electrophoresis and multilocus sequence typing results indicated four clusters, of which cluster A (n = 21, 87.5%) belonged to ST11 and the three remaining isolates (ST412, ST65, ST23) had different pulsotypes (cluster B, C, D). The blaKPC-2-carrying plasmids all belonged to IncFIIK type, and the size ranged from 100 to 390 kb. Nineteen strains (79.2%) had a 219-kb virulence plasmid possessed high similarity to pLVPK from CG43 with serotype K2. Two strains had a 224-kb virulence plasmid resembled plasmid pK2044 from K. pneumoniae NTUH-K2044(ST23). Moreover, three strains carried three different hybrid resistance- and virulence-encoding plasmids. Conjugation assays showed that both blaKPC-2 and rmpA2 genes could be successfully transferred to E. coli J53 in 62.5% of the strains at frequencies of 4.5 × 10−6 to 2.4 × 10−4, of which three co-transferred blaKPC-2 along with rmpA2 in large plasmids. Infection assays in the Galleria mellonella model demonstrated the virulence level of these isolates was found to be consistently higher than that of classic Klebsiella pneumoniae. In conclusion, CR-KP co-harboring blaKPC-2-carrying plasmid and pLVPK-like virulence plasmid were characterized by multi-drug resistance, enhanced virulence, and transferability, and should, therefore, be regarded as a real superbug that could pose a serious threat to public health. Hence, heightened efforts are urgently needed to avoid its co-transmission of the virulent plasmid (gene) and resistant plasmid (gene) in clinical isolates.

High-throughput and dosage-controlled intracellular delivery of large cargos by an acoustic-electric micro-vortices platform

ABSTRACTIntracellular delivery of cargos for cell engineering plays a pivotal role in transforming medicine and biomedical discoveries. Recent advances in microfluidics and nanotechnology have opened up new avenues for efficient, safe, and controllable intracellular delivery, as they improve precision down to the single-cell level. Based on this capability, several promising micro- and nanotechnology approaches outperform viral and conventional non-viral techniques in offering dosage-controlled delivery and/or intracellular delivery of large cargos. However, to achieve this level of precision and effectiveness, they are either low in throughput, limited to specific cell types (e.g., adherent vs. suspension cells), or complicated to operate with. To address these challenges, here we introduce a versatile and simple-to-use intracellular delivery microfluidic platform, termed Acoustic-Electric Shear Orbiting Poration (AESOP). Hundreds of acoustic microstreaming vortices form the production line of the AESOP platform, wherein hundreds of thousands of cells are trapped, permeabilized, and mixed with exogenous cargos. Using AESOP, we show intracellular delivery of a wide range of molecules (from <1 kDa to 2 MDa) with high efficiency, cell viability, and dosage-controlled capability into both suspension and adherent cells and demonstrate throughput at 1 million cells/min per single chip. In addition, we demonstrate AESOP for two gene editing applications that require delivery of large plasmids: i) eGFP plasmid (6.1 kbp) transfection, and ii) CRISPR-Cas9-mediated gene knockout using a 9.3 kbp plasmid DNA encoding Cas9 protein and sgRNA. Compared to alternative platforms, AESOP not only offers dosage-controlled intracellular delivery of large plasmids (>6kbp) with viabilities over 80% and comparable delivery efficiencies, but also is an order of magnitude higher in throughput, compatible with both adherent and suspension cell lines, and simple to operate.