Cell Membrane Adaptations Mediate β-Lactam-Induced Resensitization of Daptomycin-Resistant (DAP-R) Staphylococcus aureus In Vitro

The reversal of daptomycin resistance in MRSA to a daptomycin-susceptible phenotype following prolonged passage in selected β-lactams occurs coincident with the accumulation of multiple point mutations in the mprF gene. MprF regulates surface charge by modulating the content and translocation of the positively charged cell membrane phospholipid, lysyl-phosphatidylglycerol (LPG). The precise cell membrane adaptations accompanying such β-lactam-induced mprF perturbations are unknown. This study examined key cell membrane metrics relevant to antimicrobial resistance among three daptomycin-resistant MRSA clinical strains, which became daptomycin-susceptible following prolonged exposure to cloxacillin (‘daptomycin-resensitized’). The causal role of such secondary mprF mutations in mediating daptomycin resensitization was confirmed through allelic exchange strategies. The daptomycin-resensitized strains derived either post-cloxacillin passage or via allelic exchange (vs. their respective daptomycin-resistant strains) showed the following cell membrane changes: (i) enhanced BODIPY-DAP binding; (ii) significant reductions in LPG content, accompanied by significant increases in phosphatidylglycerol content (p < 0.05); (iii) no significant changes in positive cell surface charge; (iv) decreased cell membrane fluidity (p < 0.05); (v) enhanced carotenoid content (p < 0.05); and (vi) lower branched chain fatty acid profiles (antiso- vs. iso-), resulting in increases in saturated fatty acid composition (p < 0.05). Overall, the cell membrane characteristics of the daptomycin-resensitized strains resembled those of parental daptomycin-susceptible strains. Daptomycin resensitization with selected β-lactams results in both definable genetic changes (i.e., mprF mutations) and a number of key cell membrane phenotype modifications, which likely facilitate daptomycin activity.

Advances and Perspectives for Genome Editing Tools of Corynebacterium glutamicum

Corynebacterium glutamicum has been considered a promising synthetic biological platform for biomanufacturing and bioremediation. However, there are still some challenges in genetic manipulation of C. glutamicum. Recently, more and more genetic parts or elements (replicons, promoters, reporter genes, and selectable markers) have been mined, characterized, and applied. In addition, continuous improvement of classic molecular genetic manipulation techniques, such as allelic exchange via single/double-crossover, nuclease-mediated site-specific recombination, RecT-mediated single-chain recombination, actinophages integrase-mediated integration, and transposition mutation, has accelerated the molecular study of C. glutamicum. More importantly, emerging gene editing tools based on the CRISPR/Cas system is revolutionarily rewriting the pattern of genetic manipulation technology development for C. glutamicum, which made gene reprogramming, such as insertion, deletion, replacement, and point mutation, much more efficient and simpler. This review summarized the recent progress in molecular genetic manipulation technology development of C. glutamicum and discussed the bottlenecks and perspectives for future research of C. glutamicum as a distinctive microbial chassis.

Versatile genetic toolbox for Prevotella copri enables studying polysaccharide utilization systems

Prevotella copri is a prevalent inhabitant of the human gut and has been associated with plant-rich diet consumption and diverse health states. The underlying genetic basis of these associations remains enigmatic due to the lack of genetic tools. Here, we developed a novel versatile genetic toolbox for rapid and efficient genetic insertion and allelic exchange applicable to P. copri strains from multiple clades. Enabled by the genetic platform, we systematically investigated the specificity of polysaccharide utilization loci (PULs), and identified four highly conserved PULs for utilizing arabinan, pectic galactan, arabinoxylan and inulin, respectively. Further genetic and functional analysis of arabinan utilization systems illustrate that P. copri has evolved two distinct types of arabinan-processing PULs (PULAra) and that the type-II PULAra is significantly enriched in individuals consuming a vegan diet compared to other diets. In summary, this genetic toolbox will enable functional genetic studies for P. copri in the future.

Insertional Inactivation of Prevotella intermedia OxyR Results in Reduced Survival with Oxidative Stress and in the Presence of Host Cells

One of the most abundant bacteria in the subgingival pockets of patients with bleeding following mechanical periodontal therapy is Prevotella intermedia. However, despite its abundance, the molecular mechanisms of its contribution to periodontal disease are not well known. This is mainly due to the lack of genetic tools that would allow examination of the role of predicted virulence factors in the pathogenesis of this bacterium. Here, we report on the first mutant in the P. intermedia OMA14 strain. The mutation is an allelic exchange replacement of the sequences coding for a putative OxyR regulator with ermF sequences coding for the macrolide–lincosamide resistance in anaerobic bacteria. The mutant is severely impaired in its ability to grow with eukaryotic cells, indicating that it is an important target for interventional strategies. Further analyses reveal that its ability to grow with oxidative stress species, in the form of hydrogen peroxide and oxygen, is severely affected. Transcriptome analysis reveals that the major deregulated genes code for the alkylhydroperoxide reductase system, AhpCF, mediating protection from peroxide stress. Moreover, genes coding for Dps, CydA and Ftn are downregulated in the mutant strain, as further verified using qRT-PCR analysis. In conclusion, we succeeded in generating the first P. intermedia mutant and show that the OxyR-deficient strain is unable to survive with a variety of host cells as well as with oxidative stress.

New cloning vectors to facilitate quick allelic exchange in gram-negative bacteria

New cloning vectors have been developed with features to enhance quick allelic exchange in gram-negative bacteria. The conditionally replicative R6K and transfer origins facilitate conjugation and chromosomal integration into a variety of bacterial species, whereas the sacB gene provides counterselection for allelic exchange. The vectors have incorporated the lacZ alpha fragment with an enhanced multicloning site for easy blue/white screening and priming sites identified for efficient in vivo assembly or other DNA assembly cloning techniques. Different antibiotic resistance markers allow versatility for use with different bacteria, and transformation into an Escherichia coli strain capable of conjugation enables a quick method for allelic exchange. As a proof of principle, the authors used these vectors to inactivate genes in Vibrio cholerae and Salmonella typhimurium.

From cloning to mutant in 5 days: rapid allelic exchange in Staphylococcus aureus

In the last 10 years, the barriers preventing the uptake of foreign DNA by clinical Staphylococcus aureus isolates have been identified and powerful mutagenesis techniques such as allelic exchange are now possible in most genotypes. However, these targeted approaches can still be cumbersome, and the construction of unmarked deletions/point mutations may take many weeks or months. Here, we introduce a streamlined allelic exchange protocol using IMxxB Escherichia coli and the plasmid pIMAY-Z. With this optimized approach, a site-specific mutation can be introduced into S. aureus in 5 days, from the start of cloning to isolation of genomic DNA for confirmatory whole-genome sequencing. This streamlined protocol considerably reduces the time required to introduce a specific, unmarked mutation in S. aureus and should dramatically improve the scalability of gene-function studies.

Mass allelic exchange: enabling sexual genetics in Escherichia coli

Despite dramatic advances in genomics, connecting genotypes to phenotypes is still challenging. Sexual genetics combined with linkage analysis is a powerful solution to this problem but generally unavailable in bacteria. We build upon a strong negative selection system to invent Mass Allelic Exchange (MAE), which enables hybridization of arbitrary (including pathogenic) strains of E. coli. MAE reimplements the natural phenomenon of random crossovers, enabling classical linkage analysis. We demonstrate the utility of MAE with virulence-related gain-of-function screens, discovering that transfer of a single operon from a uropathogenic strain is sufficient for enabling a commensal E. coli to form large intracellular bacterial collections within bladder epithelial cells. MAE thus enables assaying natural allelic variation in E. coli (and potentially other bacteria), complementing existing loss-of-function genomic techniques.One Sentence SummaryWe create F1 hybrids of E. coli using MAE, bringing the power of linkage analysis to bear on phenotypic diversity (including virulence)

CRISPRi enables studies of enterococcal biofilm initiation, maturation and maintenance.

&lt;p&gt;Among the Enterococci, &lt;em&gt;Enterococcus faecalis&lt;/em&gt; is most frequently associated with human infections ranging from the urinary tract and wound infection to endocarditis and bacteraemia. These infections are often multidrug-resistant and, hence, life-threatening. Moreover, &lt;em&gt;E. faecalis&lt;/em&gt; are often co-isolated with other pathogenic bacteria from polymicrobial biofilm-associated infections contributing to disease progression and poorer patient outcomes. Genetic tools to dissect complex interactions in biofilms and mixed microbial communities are largely limited to transposon mutagenesis and traditional allelic exchange methods requiring time- and labour-intensive two-step integration and excision screening that can take a week or more to make a single mutant. We built upon the well-characterized CRISPR interference system using streptococcal dCas9 to develop an easily-modifiable, inducible system for &lt;em&gt;E. faecalis&lt;/em&gt; that can efficiently silence single and multiple genes in a matter of hours. We show that this system can silence genes involved in biofilm formation, antibiotic resistance, and can be used to interrogate gene essentiality. Uniquely, this tool is optimized to study genes important for biofilm initiation, maturation, and maintenance, and can be used to perturb pre-formed biofilms. This inducible CRISPRi system will be valuable to rapidly and efficiently investigate a wide range of aspects of complex enterococcal regulation networks within the biofilms, including polymicrobial biofilms.&amp;#160;&lt;/p&gt;

Fluorescence-Reported Allelic Exchange Mutagenesis-Mediated Gene Deletion Indicates a Requirement for Chlamydia trachomatis Tarp during In Vivo Infectivity and Reveals a Specific Role for the C Terminus during Cellular Invasion

ABSTRACT The translocated actin recruiting phosphoprotein (Tarp) is a multidomain type III secreted effector used by Chlamydia trachomatis. In aggregate, existing data suggest a role of this effector in initiating new infections. As new genetic tools began to emerge to study chlamydial genes in vivo, we speculated as to what degree Tarp function contributes to Chlamydia’s ability to parasitize mammalian host cells. To address this question, we generated a complete tarP deletion mutant using the fluorescence-reported allelic exchange mutagenesis (FRAEM) technique and complemented the mutant in trans with wild-type tarP or mutant tarP alleles engineered to harbor in-frame domain deletions. We provide evidence for the significant role of Tarp in C. trachomatis invasion of host cells. Complementation studies indicate that the C-terminal filamentous actin (F-actin)-binding domains are responsible for Tarp-mediated invasion efficiency. Wild-type C. trachomatis entry into HeLa cells resulted in host cell shape changes, whereas the tarP mutant did not. Finally, using a novel cis complementation approach, C. trachomatis lacking tarP demonstrated significant attenuation in a murine genital tract infection model. Together, these data provide definitive genetic evidence for the critical role of the Tarp F-actin-binding domains in host cell invasion and for the Tarp effector as a bona fide C. trachomatis virulence factor.