Complete genome sequence of Pseudomonas aeruginosa Y010, a taro rhizosphere strain producing potent antimicrobial agents.

Pseudomonas aeruginosa Y010, isolated from the taro rhizosphere, exhibits great antagonistic abilities against Dickeya strains that cause soft-rot and blackleg diseases of plants by producing potent antimicrobial agents. The complete genome of Y010 was sequenced and annotated, which is 6,415,628 bp in length with 66.39% GC content.

A cyclic di-GMP-binding adaptor protein interacts with a N5-glutamine methyltransferase to regulate the pathogenesis in Xanthomonas citri subsp. citri.

The second messenger cyclic diguanylate monophosphate (c-di-GMP) regulates a wide range of bacterial behaviours through diverse mechanisms and binding receptors. Single-domain PilZ proteins, the most widespread and abundant known c-di-GMP receptors in bacteria, act as trans-acting adaptor proteins that enable c-di-GMP to control signalling pathways with high specificity. This study identifies a single-domain PilZ protein, XAC3402 (renamed N5MapZ), from the phytopathogen Xanthomonas citri subsp. citri (Xcc), which modulates Xcc virulence by directly interacting with the methyltransferase HemK. Through yeast two-hybrid, co-immunoprecipitation and immunofluorescent staining, we demonstrated that N5MapZ and HemK interact directly under both in vitro and in vivo conditions, with the strength of the protein-protein interaction decreasing at high c-di-GMP concentrations. This finding distinguishes N5MapZ from other characterized single-domain PilZ proteins, as it was previously known that c-di-GMP enhances the interaction between those single-domain PilZs and their protein partners. This observation is further supported by the fact that the c-di-GMP binding-defective mutant N5MapZR10A can interact with HemK to inhibit the methylation of the class 1 translation termination release factor PrfA. Additionally, we found that HemK plays an important role in Xcc pathogenesis, as the deletion of hemK leads to extensive phenotypic changes, including reduced virulence in citrus plants, decreased motility, production of extracellular enzymes and stress tolerance. Gene expression analysis has revealed that c-di-GMP and the HemK-mediated pathway regulate the expression of multiple virulence effector proteins, uncovering a novel regulatory mechanism through which c-di-GMP regulates Xcc virulence by mediating PrfA methylation via the single-domain PilZ adaptor protein N5MapZ.

Native CRISPR-Cas-based programmable multiplex gene repression in Klebsiella variicola.

Klebsiella variicola is a Gram-negative bacterium that is frequently isolated from a wide variety of natural niches. It is a ubiquitous opportunistic pathogen that can cause diverse infections in plants, animals, and humans. It also has significant biotechnological potential. However, due to the lack of efficient genetic tools, the molecular basis contributing to the pathogenesis and beneficial activities of K. variicola remains poorly understood. In this study, we found and characterized a native type I-E CRISPR-Cas system in a recently isolated K. variicola strain KV-1. The system cannot cleave target DNA sequences due to the inactivation of the Cas3 nuclease by a transposable element but retains the activity of the crRNA-guided Cascade binding to the target DNA sequence. A targeting plasmid carrying a mini-CRISPR to encode a crRNA was designed and introduced into the KV-1 strain, which successfully repurposed the native type I-E CRISPR-Cas system to inhibit the expression of the target gene efficiently and specifically. Moreover, by creating a mini-CRISPR to encode multiple crRNAs, multiplex gene repression was achieved by providing a single targeting plasmid. This work provides the first native CRISPR-Cas-based tool for programmable multiplex gene repression in K. variicola, which will facilitate studying the pathogenic mechanism of K. variicola and enable metabolic engineering to produce valuable bioproducts.

Metal-regulated antibiotic resistance and its implications for antibiotic therapy.

Antibiotic resistance, one of the major medical threats worldwide, can be selected and induced by metals through multiple mechanisms such as co-resistance, cross-resistance, and co-regulation. Compared with co-resistance and cross-resistance which are attributed to the physically or functionally linked metal and antibiotic resistance genes, co-regulation of antibiotic resistance genes by metal-responsive regulators and pathways is much more complex and elusive. Here, we discussed the main mechanisms by which antibiotic resistance is regulated in response to metals and showed recent attempts to combat antibiotic resistance by interfering with metal-based signalling pathways. Further efforts to depict the intricate metal-based regulatory network of antibiotic resistance will provide tremendous opportunities for the discovery of novel anti-resistance targets, and blocking or rewiring the metal-based signalling pathways is emerging as a promising stratagem to reverse bacterial resistance to antibiotics and rejuvenate the efficacy of conventional antibiotics.

Antibiotic influx and efflux in Pseudomonas aeruginosa: Regulation and therapeutic implications.

Pseudomonas aeruginosa is a notorious multidrug-resistant pathogen that poses a serious and growing threat to the worldwide public health. The expression of resistance determinants is exquisitely modulated by the abundant regulatory proteins and the intricate signal sensing and transduction systems in this pathogen. Downregulation of antibiotic influx porin proteins and upregulation of antibiotic efflux pump systems owing to mutational changes in their regulators or the presence of distinct inducing molecular signals represent two of the most efficient mechanisms that restrict intracellular antibiotic accumulation and enable P. aeruginosa to resist multiple antibiotics. Treatment of P. aeruginosa infections is extremely challenging due to the highly inducible mechanism of antibiotic resistance. This review comprehensively summarizes the regulatory networks of the major porin proteins (OprD and OprH) and efflux pumps (MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY) that play critical roles in antibiotic influx and efflux in P. aeruginosa. It also discusses promising therapeutic approaches using safe and efficient adjuvants to enhance the efficacy of conventional antibiotics to combat multidrug-resistant P. aeruginosa by controlling the expression levels of porins and efflux pumps. This review not only highlights the complexity of the regulatory network that induces antibiotic resistance in P. aeruginosa but also provides important therapeutic implications in targeting the inducible mechanism of resistance.

Suppression of Pseudomonas aeruginosa type III secretion system by a novel calcium-responsive signaling pathway.

Expression of the type III secretion system (T3SS) in Pseudomonas aeruginosa is exquisitely controlled by diverse environmental or host-related signals such as calcium (Ca2+), however, the signal transduction pathways remain largely elusive. In this study, we reported that FleR, the response regulator of the two-component system FleS/FleR, inhibits T3SS gene expression and virulence of P. aeruginosa uncoupled from its cognate histidine kinase FleS. Interestingly, FleR was found to repress T3SS gene expression under Ca2+-rich conditions independently of its DNA-binding domain. FleR activates the elevation of intracellular c-di-GMP contents and FleQ serves as the c-di-GMP effector to repress T3SS gene expression through the Gac/Rsm pathway. Remarkably, we found that AmrZ, a member of the FleR regulon, inhibits T3SS gene expression by directly targeting the promoter of exsCEBA in an expression level-dependent manner. This study revealed an intricate regulatory network that connects P. aeruginosa T3SS gene expression to the Ca2+ signal.

Promoter regulatory mode evolution enhances the high multidrug resistance of tmexCD1-toprJ1.

Antibiotic resistance could rapidly emerge from acquiring the mobile antibiotic resistance genes, which are commonly evolved from an intrinsic gene. The emergence of the plasmid-borne mobilized efflux pump gene cluster tmexCD1-toprJ1 renders the last-resort antibiotic tigecycline ineffective, although its evolutionary mechanism remains unclear. In this study, we investigate the regulatory mechanisms of the progenitor NfxB-MexCD-OprJ, a chromosomally encoded operon that does not mediate antibiotic resistance in the wild-type version, and its homologs, TNfxB1-TMexCD1-TOprJ1 mediating high-level tigecycline resistance, and TNfxB3-TMexCD3-TOprJ1. Mechanistic studies demonstrated that in nfxB-mexCD-oprJ, MexCD expression was under a weaker promoter, PmexC and inhibited by a strong repressor NfxB. For tmexCD1-toprJ1, TMexCD1 was highly expressed owing to the presence of a strong promoter, PtmexC1, and an inactive suppressor, TNfxB1, with a T39R mutation that rendered it unable to bind to promoter DNA. In tnfxB3-tmexCD3-toprJ1b, TMexCD3 expression was intermediate because of the local regulator TNfxB3, which binds to two inverted repeat sequences of PtmexC. Additionally, TNfxB3 exhibited lower protein expression and weaker DNA binding affinity than its ancestor NfxB, together with their promoter activities difference explaining the different expression levels of tmexCD-toprJ homologs. Distinct fitness burdens on these homologs-carrying bacteria were observed due to the corresponding expression level, which might be associated with their global prevalence. In summary, our data depict the mechanisms underlying the evolution and dissemination of an important mobile antibiotic resistance gene from an intrinsic chromosomal gene.IMPORTANCEAs antibiotic resistance seriously challenges global health, tigecycline is one of the few effective drugs in the pipeline against infections caused by multidrug-resistant pathogens. Our previous work identified a novel tigecycline resistance efflux pump gene cluster tmexCD1-toprJ1 in animals and humans, together with its various variants, a rising clinical concern. Herein, this study focused on how the local regulation modes of tmexCD1-toprJ1 evolved to a highly expressed efflux pump. Through comparative analysis between three tnfxB-tmexCD-toprJ homologs and their progenitor nfxB-mexCD-oprJ, modes, we demonstrated the evolutionary dynamics from a chromosomal silent gene to an active state. We found the de-repression of the local regulator and an increase of the promoter activity work together to promote a high production of drug efflux machines and enhance multidrug resistance. Our findings revealed that TMexCD1-TOprJ1 adopts a distinct evolutionary path to achieve higher multidrug resistance, urgently needing tight surveillance.

Activation of CzcS/CzcR during zinc excess regulates copper tolerance and pyochelin biosynthesis of Pseudomonas aeruginosa.

Zinc is an important transition metal that is essential for numerous physiological processes while excessive zinc is cytotoxic. Pseudomonas aeruginosa is a ubiquitous opportunistic human pathogen equipped with an exquisite zinc homeostatic system, and the two-component system CzcS/CzcR plays a key role in zinc detoxification. Although an increasing number of studies have shown the versatility of CzcS/CzcR, its physiological functions are still not fully understood. In this study, transcriptome analysis was performed, which revealed that CzcS/CzcR is silenced in the absence of the zinc signal but modulates global gene expression when the pathogen encounters zinc excess. CzcR was demonstrated to positively regulate the copper tolerance gene ptrA and negatively regulate the pyochelin biosynthesis regulatory gene pchR through direct binding to their promoters. Remarkably, the upregulation of ptrA and downregulation of pchR were shown to rescue the impaired capacity of copper tolerance and prevent pyochelin overproduction, respectively, caused by zinc excess. This study not only advances our understanding of the regulatory spectrum of CzcS/CzcR but also provides new insights into stress adaptation mediated by two-component systems in bacteria to balance the cellular processes that are disturbed by their signals. IMPORTANCE: CzcS/CzcR is a two-component system that has been found to modulate zinc homeostasis, quorum sensing, and antibiotic resistance in Pseudomonas aeruginosa. To fully understand the physiological functions of CzcS/CzcR, we performed a comparative transcriptome analysis in this study and discovered that CzcS/CzcR controls global gene expression when it is activated during zinc excess. In particular, we demonstrated that CzcS/CzcR is critical for maintaining copper tolerance and iron homeostasis, which are disrupted during zinc excess, by inducing the expression of the copper tolerance gene ptrA and repressing the pyochelin biosynthesis genes through pchR. This study revealed the global regulatory functions of CzcS/CzcR and described a new and intricate adaptive mechanism in response to zinc excess in P. aeruginosa. The findings of this study have important implications for novel anti-infective interventions by incorporating metal-based drugs.

Type I CRISPR-Cas-mediated microbial gene editing and regulation.

There are six major types of CRISPR-Cas systems that provide adaptive immunity in bacteria and archaea against invasive genetic elements. The discovery of CRISPR-Cas systems has revolutionized the field of genetics in many organisms. In the past few years, exploitations of the most abundant class 1 type I CRISPR-Cas systems have revealed their great potential and distinct advantages to achieve gene editing and regulation in diverse microorganisms in spite of their complicated structures. The widespread and diversified type I CRISPR-Cas systems are becoming increasingly attractive for the development of new biotechnological tools, especially in genetically recalcitrant microbial strains. In this review article, we comprehensively summarize recent advancements in microbial gene editing and regulation by utilizing type I CRISPR-Cas systems. Importantly, to expand the microbial host range of type I CRISPR-Cas-based applications, these structurally complicated systems have been improved as transferable gene-editing tools with efficient delivery methods for stable expression of CRISPR-Cas elements, as well as convenient gene-regulation tools with the prevention of DNA cleavage by obviating deletion or mutation of the Cas3 nuclease. We envision that type I CRISPR-Cas systems will largely expand the biotechnological toolbox for microbes with medical, environmental and industrial importance.