A mouse DRG genetic toolkit reveals morphological and physiological diversity of somatosensory neuron subtypes.

Dorsal root ganglia (DRG) somatosensory neurons detect mechanical, thermal, and chemical stimuli acting on the body. Achieving a holistic view of how different DRG neuron subtypes relay neural signals from the periphery to the CNS has been challenging with existing tools. Here, we develop and curate a mouse genetic toolkit that allows for interrogating the properties and functions of distinct cutaneous targeting DRG neuron subtypes. These tools have enabled a broad morphological analysis, which revealed distinct cutaneous axon arborization areas and branching patterns of the transcriptionally distinct DRG neuron subtypes. Moreover, in vivo physiological analysis revealed that each subtype has a distinct threshold and range of responses to mechanical and/or thermal stimuli. These findings support a model in which morphologically and physiologically distinct cutaneous DRG sensory neuron subtypes tile mechanical and thermal stimulus space to collectively encode a wide range of natural stimuli.

A cell-free transcription-translation pipeline for recreating methylation patterns boosts DNA transformation in bacteria.

The bacterial world offers diverse strains for understanding medical and environmental processes and for engineering synthetic biological chassis. However, genetically manipulating these strains has faced a long-standing bottleneck: how to efficiently transform DNA. Here, we report imitating methylation patterns rapidly in TXTL (IMPRINT), a generalized, rapid, and scalable approach based on cell-free transcription-translation (TXTL) to overcome DNA restriction, a prominent barrier to transformation. IMPRINT utilizes TXTL to express DNA methyltransferases from a bacterium's restriction-modification systems. The expressed methyltransferases then methylate DNA in vitro to match the bacterium's DNA methylation pattern, circumventing restriction and enhancing transformation. With IMPRINT, we efficiently multiplex methylation by diverse DNA methyltransferases and enhance plasmid transformation in gram-negative and gram-positive bacteria. We also develop a high-throughput pipeline that identifies the most consequential methyltransferases, and we apply IMPRINT to screen a ribosome-binding site library in a hard-to-transform Bifidobacterium. Overall, IMPRINT can enhance DNA transformation, enabling the use of sophisticated genetic manipulation tools across the bacterial world.

Genomics Reveals Complex Population History and Unexpected Diversity of Eurasian Otters (Lutra lutra) in Britain Relative to Genetic Methods.

Conservation genetic analyses of many endangered species have been based on genotyping of microsatellite loci and sequencing of short fragments of mtDNA. The increase in power and resolution afforded by whole genome approaches may challenge conclusions made on limited numbers of loci and maternally inherited haploid markers. Here, we provide a matched comparison of whole genome sequencing versus microsatellite and control region (CR) genotyping for Eurasian otters (Lutra lutra). Previous work identified four genetically differentiated "stronghold" populations of otter in Britain, derived from regional populations that survived the population crash of the 1950s-1980s. Using whole genome resequencing data from 45 samples from across the British stronghold populations, we confirmed some aspects of population structure derived from previous marker-driven studies. Importantly, we showed that genomic signals of the population crash bottlenecks matched evidence from otter population surveys. Unexpectedly, two strongly divergent mitochondrial lineages were identified that were undetectable using CR fragments, and otters in the east of England were genetically distinct and surprisingly variable. We hypothesize that this previously unsuspected variability may derive from past releases of Eurasian otters from other, non-British source populations in England around the time of the population bottleneck. Our work highlights that even reasonably well-studied species may harbor genetic surprises, if studied using modern high-throughput sequencing methods.

Teaching old dogs new tricks: genetic engineering methanogens.

Methanogenic archaea, which are integral to global carbon and nitrogen cycling, currently face challenges in genetic manipulation due to unique physiology and limited genetic tools. This review provides a survey of current and past developments in the genetic engineering of methanogens, including selection and counterselection markers, reporter systems, shuttle vectors, mutagenesis methods, markerless genetic exchange, and gene expression control. This review discusses genetic tools and emphasizes challenges tied to tool scarcity for specific methanogenic species. Mutagenesis techniques for methanogens, including physicochemical, transposon-mediated, liposome-mediated mutagenesis, and natural transformation, are outlined, along with achievements and challenges. Markerless genetic exchange strategies, such as homologous recombination and CRISPR/Cas-mediated genome editing, are also detailed. Finally, the review concludes by examining the control of gene expression in methanogens. The information presented underscores the urgent need for refined genetic tools in archaeal research. Despite historical challenges, recent advancements, notably CRISPR-based systems, hold promise for overcoming obstacles, with implications for global health, agriculture, climate change, and environmental engineering. This comprehensive review aims to bridge existing gaps in the literature, guiding future research in the expanding field of archaeal genetic engineering.

A CRISPR-Cas-associated transposon system for genome editing in Burkholderia cepacia complex species.

Genome editing in non-model bacteria is important to understand gene-to-function links that may differ from those of model microorganisms. Although species of the Burkholderia cepacia complex (Bcc) have great biotechnological capacities, the limited genetic tools available to understand and mitigate their pathogenic potential hamper their utilization in industrial applications. To broaden the genetic tools available for Bcc species, we developed RhaCAST, a targeted DNA insertion platform based on a CRISPR-associated transposase driven by a rhamnose-inducible promoter. We demonstrated the utility of the system for targeted insertional mutagenesis in the Bcc strains B. cenocepacia K56-2 and Burkholderia multivorans ATCC17616. We showed that the RhaCAST system can be used for loss- and gain-of-function applications. Importantly, the selection marker could be excised and reused to allow iterative genetic manipulation. The RhaCAST system is faster, easier, and more adaptable than previous insertional mutagenesis tools available for Bcc species and may be used to disrupt pathogenicity elements and insert relevant genetic modules, enabling Bcc biotechnological applications. IMPORTANCE: Species of the Burkholderia cepacia complex (Bcc) have great biotechnological potential but are also opportunistic pathogens. Genetic manipulation of Bcc species is necessary to understand gene-to-function links. However, limited genetic tools are available to manipulate Bcc, hindering our understanding of their pathogenic traits and their potential in biotechnological applications. We developed a genetic tool based on CRISPR-associated transposase to increase the genetic tools available for Bcc species. The genetic tool we developed in this study can be used for loss and gain of function in Bcc species. The significance of our work is in expanding currently available tools to manipulate Bcc.

Metabolic engineering strategies to enable microbial electrosynthesis utilization of CO2: recent progress and challenges.

Microbial electrosynthesis (MES) is a promising technology that mainly utilizes microbial cells to convert CO2 into value-added chemicals using electrons provided by the cathode. However, the low electron transfer rate is a solid bottleneck hindering the further application of MES. Thus, as an effective strategy, genetic tools play a key role in MES for enhancing the electron transfer rate and diversity of production. We describe a set of genetic strategies based on fundamental characteristics and current successes and discuss their functional mechanisms in driving microbial electrocatalytic reactions to fully comprehend the roles and uses of genetic tools in MES. This paper also analyzes the process of nanomaterial application in extracellular electron transfer (EET). It provides a technique that combines nanomaterials and genetic tools to increase MES efficiency, because nanoparticles have a role in the production of functional genes in EET although genetic tools can subvert MES, it still has issues with difficult transformation and low expression levels. Genetic tools remain one of the most promising future strategies for advancing the MES process despite these challenges.

Identifying functional multi-host shuttle plasmids to advance synthetic biology applications in Mesorhizobium and Bradyrhizobium.

Ammonia availability has a crucial role in agriculture as it ensures healthy plant growth and increased crop yields. Since diazotrophs are the only organisms capable of reducing dinitrogen to ammonia, they have great ecological importance and potential to mitigate the environmental and economic costs of synthetic fertilizer use. Rhizobia are especially valuable being that they can engage in nitrogen-fixing symbiotic relationships with legumes, and they demonstrate great diversity and plasticity in genomic and phenotypic traits. However, few rhizobial species have sufficient genetic tractability for synthetic biology applications. This study established a basic genetic toolbox with antibiotic resistance markers, multi-host shuttle plasmids and a streamlined protocol for biparental conjugation with Mesorhizobium and Bradyrhizobium species. We identified two repABC origins of replication from Sinorhizobium meliloti (pSymB) and Rhizobium etli (p42d) that were stable across all three strains of interest. Furthermore, the NZP2235 genome was sequenced and phylogenetic analysis determined its reclassification to Mesorhizobium huakuii. These tools will enable the use of plasmid-based strategies for more advanced genetic engineering projects and ultimately contribute towards the development of more sustainable agriculture practices by means of novel nitrogen-fixing organelles, elite bioinoculants, or symbiotic association with nonlegumes.

Isolation of a facultative methanotroph Methylocystis iwaonis SD4 from rice rhizosphere and establishment of rapid genetic tools for it.

Methanotrophs of the genus Methylocystis are frequently found in rice paddies. Although more than ten facultative methanotrophs have been reported since 2005, none of these strains was isolated from paddy soil. Here, a facultative methane-oxidizing bacterium, Methylocystis iwaonis SD4, was isolated and characterized from rhizosphere samples of rice plants in Nanjing, China. This strain grew well on methane or methanol but was able to grow slowly using acetate or ethanol. Moreover, strain SD4 showed sustained growth at low concentrations of methane (100 and 500 ppmv). M. iwaonis SD4 could utilize diverse nitrogen sources, including nitrate, urea, ammonium as well as dinitrogen. Strain SD4 possessed genes encoding both the particulate methane monooxygenase and the soluble methane monooxygenase. Simple and rapid genetic manipulation methods were established for this strain, enabling vector transformation and unmarked genetic manipulation. Fast growth rate and efficient genetic tools make M. iwaonis SD4 an ideal model to study facultative methanotrophs, and the ability to grow on low concentration of methane implies its potential in methane removal.

Inducible CRISPRi-Based Operon Silencing and Selective in Trans Gene Complementation in Borrelia burgdorferi.

To accelerate genetic studies on the Lyme disease pathogen Borrelia burgdorferi, we developed an enhanced CRISPR interference (CRISPRi) approach for isopropyl-ß-d-thiogalactopyranoside (IPTG)-inducible repression of specific B. burgdorferi genes. The entire system is encoded on a compact 11-kb shuttle vector plasmid that allows for inducible expression of both the sgRNA module and a nontoxic codon-optimized dCas9 protein. We validated this CRISPRi system by targeting the genes encoding OspA and OspB, abundant surface lipoproteins coexpressed by a single operon, and FlaB, the major subunit forming the periplasmic flagella. As in other systems, single guide RNAs (sgRNAs) complementary to the nontemplate strand were consistently effective in gene repression, with 4- to 994-fold reductions in targeted transcript levels and concomitant reductions of protein levels. Furthermore, we showed that ospAB knockdowns could be selectively complemented in trans for OspA expression via the insertion of CRISPRi-resistant, synonymously or nonsynonymously mutated protospacer adjacent motif (PAM*) ospA alleles into a unique site within the CRISPRi plasmid. Together, this establishes CRISPRi PAM* as a robust new genetic tool to simplify the study of B. burgdorferi genes, bypassing the need for gene disruptions by allelic exchange and avoiding rare codon toxicity from the heterologous expression of dCas9. IMPORTANCE Borrelia burgdorferi, the spirochetal bacterium causing Lyme disease, is a tick-borne pathogen of global importance. Here, we expand the genetic toolbox for studying B. burgdorferi physiology and pathogenesis by establishing a single plasmid-based, fully inducible, and nontoxic CRISPR interference (CRISPRi) system for transcriptional silencing of B. burgdorferi genes and operons. We also show that alleles of CRISPRi-targeted genes with mutated protospacer-adjacent motif (PAM*) sites are CRISPRi resistant and can be used for simultaneous in trans gene complementation. The CRISPRi PAM* system will streamline the study of essential Borrelia proteins and accelerate investigations into their structure-function relationships.

Monarch Butterfly Migration Moving into the Genetic Era.

The genetic architecture and neurogenetics of animal migration remain poorly understood. With a sequenced genome and the establishment of reverse genetic tools, the monarch butterfly has emerged as a promising model to uncover the genetic basis of migratory behavior and associated traits. Here, we synthesize major advances made in the genetics of monarch migration, which includes the discovery of genomic regions associated with migration and molecular mechanisms underpinning its seasonality. We highlight the catalytic role that a rapidly growing number of contemporary genetic and molecular technologies applicable to nonconventional organisms have had in these discoveries, and outline new avenues of investigation to continue moving the field forward.

A genetic system for tissue-specific inhibition of cell proliferation.

Cellular proliferation is a basic process during organ development, tissue homeostasis and disease progression. Likewise, after injury typically multiple cell lineages respond to various cues and proliferate to initiate repair and/or remodeling of the injured tissue. Unravelling the specific role of proliferation of one cell type and its lineage in the context of the whole organism during tissue regeneration and/or disease progression would provide valuable information on these processes. Here, we report a new genetic system that allows cell proliferation to be inhibited in a tissue-specific manner. We generated Cre- or Dre-inducible p21-GFP (ip21-GFP) transgenic mice that enable experimentally induced permanent cell cycle arrest of specific cell lineages of interest, while genetically marking these cells. This system allows for the inhibition of pathogenic cell proliferation. We found that cardiac fibroblast proliferation inhibition significantly reduced scar formation, and promoted neovascularization and cardiomyocyte survival. Additionally, we found that inhibition of one type of cell proliferation (namely, hepatocytes) induces the lineage conversion of another type cells (i.e. ductal cells) during tissue regeneration. These results validate the use of ip21-GFP mice as a new genetic tool for cell lineage-specific inhibition of cell proliferation in vivo.

A Synthetic Cumate-Inducible Promoter for Graded and Homogenous Gene Expression in Pseudomonas aeruginosa.

Inducible gene expression systems are powerful genetic tools to study bacterial physiology, probing essential and toxic gene functions, gene dosage effects, and overexpression phenotypes. For the opportunistic human pathogen Pseudomonas aeruginosa, dedicated inducible gene expression systems are scarce. In the current study, we developed a minimal synthetic 4-isopropylbenzoic acid (cumate)-inducible promoter, called PQJ, that is tunable over several orders of magnitude. This was achieved by combining semirandomized housekeeping promoter libraries and control elements from the Pseudomonas putida strain F1 cym/cmt system with powerful fluorescence-activated cell sorting (FACS) to select functionally optimized variants. Using flow cytometry and live-cell fluorescence microscopy, we demonstrate that PQJ responds rapidly and homogenously to the inducer cumate in a graded manner at the single-cell level. PQJ and cumate are orthogonal to the frequently used isopropyl ß-d-thiogalactopyranoside (IPTG)-regulated lacIq-Ptac expression system. The modular design of the cumate-inducible expression cassette together with the FACS-based enrichment strategy presented here facilitates portability, thus serving as a blueprint for the development of tailored gene expression systems for a wide range of bacteria. IMPORTANCE Reverse genetics is a powerful approach to study bacterial physiology and behavior by relying on well-developed genetic tools, such as inducible promoters. For the human pathogen Pseudomonas aeruginosa, well-characterized inducible promoters are scarce. In the current work, we used a synthetic biology-based approach to develop a cumate-inducible promoter for P. aeruginosa, termed PQJ, that shows excellent induction properties at the single-cell level. This genetic tool provides the means for qualitative and quantitative gene function studies describing P. aeruginosa's physiology and virulence in vitro and in vivo. Because this synthetic approach to constructing species-specific inducible promoters is portable, it can serve as a blueprint for similar tailored gene expression systems in bacteria largely lacking such tools, including, for example, representatives of the human microbiota.

Model systems for regeneration: zebrafish.

Tissue damage can resolve completely through healing and regeneration, or can produce permanent scarring and loss of function. The response to tissue damage varies across tissues and between species. Determining the natural mechanisms behind regeneration in model organisms that regenerate well can help us develop strategies for tissue recovery in species with poor regenerative capacity (such as humans). The zebrafish (Danio rerio) is one of the most accessible vertebrate models to study regeneration. In this Primer, we highlight the tools available to study regeneration in the zebrafish, provide an overview of the mechanisms underlying regeneration in this system and discuss future perspectives for the field.

Research Priorities for Kidney-Related Research-An Agenda to Advance Kidney Care: A Position Statement From the National Kidney Foundation.

Despite the high prevalence and economic burden of chronic kidney disease (CKD) in the United States, federal funding for kidney-related research, prevention, and education activities under the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) remains substantially lower compared to other chronic diseases. More federal support is needed to promote critical research that will expand knowledge of kidney health and disease, develop new and effective therapies, and reduce health disparities. In 2021, the National Kidney Foundation (NKF) convened 2 Research Roundtables (preclinical and clinical research), comprising nephrology leaders from prominent US academic institutions and the pharmaceutical industry, key bodies with expertise in research, and including individuals with CKD and their caregivers and kidney donors. The goal of these roundtables was to identify priorities for preclinical and clinical kidney-related research. The research priorities identified by the Research Roundtables and presented in this position statement outline attainable opportunities for groundbreaking and critically needed innovations that will benefit patients with kidney disease in the next 5-10 years. Research priorities fall within 4 preclinical science themes (expand data science capability, define kidney disease mechanisms and utilize genetic tools to identify new therapeutic targets, develop better models of human disease, and test cell-specific drug delivery systems and utilize gene editing) and 3 clinical science themes (expand number and inclusivity of clinical trials, develop and test interventions to reduce health disparities, and support implementation science). These priorities in kidney-related research, if supported by additional funding by federal agencies, will increase our understanding of the development and progression of kidney disease among diverse populations, attract additional industry investment, and lead to new and more personalized treatments.

SEVA-Cpf1, a CRISPR-Cas12a vector for genome editing in cyanobacteria.

BACKGROUND: Cyanobacteria are photosynthetic autotrophs that have tremendous potential for fundamental research and industrial applications due to their high metabolic plasticity and ability to grow using CO2 and sunlight. CRISPR technology using Cas9 and Cpf1 has been applied to different cyanobacteria for genome manipulations and metabolic engineering. Despite significant advances with genome editing in several cyanobacteria strains, the lack of proper genetic toolboxes is still a limiting factor compared to other model laboratory species. Among the limitations, it is essential to have versatile plasmids that could ease the benchwork when using CRISPR technology. RESULTS: In the present study, several CRISPR-Cpf1 vectors were developed for genetic manipulations in cyanobacteria using SEVA plasmids. SEVA collection is based on modular vectors that enable the exchangeability of diverse elements (e.g. origins of replication and antibiotic selection markers) and the combination with many cargo sequences for varied end-applications. Firstly, using SEVA vectors containing the broad host range RSF1010 origin we demonstrated that these vectors are replicative not only in model cyanobacteria but also in a new cyanobacterium specie, Chroococcidiopsis sp., which is different from those previously published. Then, we constructed SEVA vectors by harbouring CRISPR elements and showed that they can be easily assimilated not only by conjugation, but also by natural transformation. Finally, we used our SEVA-Cpf1 tools to delete the nblA gene in Synechocystis sp. PCC 6803, demonstrating that our plasmids can be applied for CRISPR-based genome editing technology. CONCLUSIONS: The results of this study provide new CRISPR-based vectors based on the SEVA (Standard European Vector Architecture) collection that can improve editing processes using the Cpf1 nuclease in cyanobacteria.

Development and application of CRISPR-based genetic tools in Bacillus species and Bacillus phages.

Recently, the clustered regularly interspaced short palindromic repeats (CRISPR) system has been developed into a precise and efficient genome editing tool. Since its discovery as an adaptive immune system in prokaryotes, it has been applied in many different research fields including biotechnology and medical sciences. The high demand for rapid, highly efficient and versatile genetic tools to thrive in bacteria-based cell factories accelerates this process. This review mainly focuses on significant advancements of the CRISPR system in Bacillus subtilis, including the achievements in gene editing, and on problems still remaining. Next, we comprehensively summarize this genetic tool's up-to-date development and utilization in other Bacillus species, including B. licheniformis, B. methanolicus, B. anthracis, B. cereus, B. smithii and B. thuringiensis. Furthermore, we describe the current application of CRISPR tools in phages to increase Bacillus hosts' resistance to virulent phages and phage genetic modification. Finally, we suggest potential strategies to further improve this advanced technique and provide insights into future directions of CRISPR technologies for rendering Bacillus species cell factories more effective and more powerful.

Invited review: Unearthing the mechanisms of age-related neurodegenerative disease using Caenorhabditis elegans.

As human life expectancy increases, neurodegenerative diseases present a growing public health threat, for which there are currently few effective treatments. There is an urgent need to understand the molecular and genetic underpinnings of these disorders so new therapeutic targets can be identified. Here we present the argument that the simple nematode worm Caenorhabditis elegans is a powerful tool to rapidly study neurodegenerative disorders due to their short lifespan and vast array of genetic tools, which can be combined with characterization of conserved neuronal processes and behavior orthologous to those disrupted in human disease. We review how pre-existing C. elegans models provide insight into human neurological disease as well as an overview of current tools available to study neurodegenerative diseases in the worm, with an emphasis on genetics and behavior. We also discuss open questions that C. elegans may be particularly well suited for in future studies and how worms will be a valuable preclinical model to better understand these devastating neurological disorders.

A Homologous Recombination System to Generate Epitope-Tagged Target Genes in Chaetomium thermophilum: A Genetic Approach to Investigate Native Thermostable Proteins.

Chaetomium thermophilum is an attractive eukaryotic model organism which, due to its unusually high temperature tolerance (optimal growth at 50-52 °C), has a thermostable proteome that can be exploited for biochemical, structural and biotechnological applications. Site directed gene manipulation for the expression of labeled target genes is a desirable approach to study the structure and function of thermostable proteins and their organization in complexes, which has not been established for this thermophile yet. Here, we describe the development of a homologous recombination system to epitope-tag chromosomal genes of interest in Chaetomium thermophilum with the goal to exploit the derived thermostable fusion proteins for tandem-affinity purification. This genetic approach was facilitated by the engineering of suitable strains, in which factors of the non-homologous end-joining pathway were deleted, thereby improving the efficiency of homologous integration at specific gene loci. Following this strategy, we could demonstrate that gene tagging via homologous recombination improved the yield of purified bait proteins and co-precipitated factors, paving the way for related studies in fundamental research and industrial applications.

It takes a neural village: Circuit-based approaches for estrogenic regulation of episodic memory.

Cognitive behaviors, such as episodic memory formation, are complex processes involving coordinated activity in multiple brain regions. However, much of the research on hormonal regulation of cognition focuses on manipulation of one region at a time or provides a single snapshot of how a systemic treatment affects multiple brain regions without investigating how these regions might interact to mediate hormone effects. Here, we use estrogenic regulation of episodic memory as an example of how circuit-based approaches may be incorporated into future studies of hormones and cognition. We first review basic episodic memory circuitry, rapid mechanisms by which 17ß-estradiol can alter circuit activity, and current knowledge about 17ß-estradiol's effects on episodic memory. Next, we outline approaches that researchers can employ to consider circuit effects in their estrogen research and provide examples of how these methods have been used to examine hormonal regulation of memory and other behaviors.

Generation of Genetic Tools for Gauging Multiple-Gene Expression at the Single-Cell Level.

Key microbial processes in many bacterial species are heterogeneously expressed in single cells of bacterial populations. However, the paucity of adequate molecular tools for live, real-time monitoring of multiple-gene expression at the single-cell level has limited the understanding of phenotypic heterogeneity. To investigate phenotypic heterogeneity in the ubiquitous opportunistic pathogen Pseudomonas aeruginosa, a genetic tool that allows gauging multiple-gene expression at the single-cell level has been generated. This tool, named pRGC, consists of a promoter-probe vector for transcriptional fusions that carries three reporter genes coding for the fluorescent proteins mCherry, green fluorescent protein (GFP), and cyan fluorescent protein (CFP). The pRGC vector has been characterized and validated via single-cell gene expression analysis of both constitutive and iron-regulated promoters, showing clear discrimination of the three fluorescence signals in single cells of a P. aeruginosa population without the need for image processing for spectral cross talk correction. In addition, two pRGC variants have been generated for either (i) integration of the reporter gene cassette into a single neutral site of P. aeruginosa chromosome that is suitable for long-term experiments in the absence of antibiotic selection or (ii) replication in bacterial genera other than Pseudomonas The easy-to-use genetic tools generated in this study will allow rapid and cost-effective investigation of multiple-gene expression in populations of environmental and pathogenic bacteria, hopefully advancing the understanding of microbial phenotypic heterogeneity.IMPORTANCE Within a bacterial population, single cells can differently express some genes, even though they are genetically identical and experience the same chemical and physical stimuli. This phenomenon, known as phenotypic heterogeneity, is mainly driven by gene expression noise and results in the emergence of bacterial subpopulations with distinct phenotypes. The analysis of gene expression at the single-cell level has shown that phenotypic heterogeneity is associated with key bacterial processes, including competence, sporulation, and persistence. In this study, new genetic tools have been generated that allow easy cloning of up to three promoters upstream of distinct fluorescent genes, making it possible to gauge multiple-gene expression at the single-cell level by fluorescence microscopy without the need for advanced image-processing procedures. A proof of concept has been provided by investigating iron uptake and iron storage gene expression in response to iron availability in P. aeruginosa.