A novel and quick PCR-based method to genotype mice with a leptin receptor mutation (db/db mice).

db/db mice is one of most widely used animal models in studying the cellular and molecular mechanisms of metabolic disorders, such as diabetes, hyperlipidemia, and obesity. The mice carry spontaneous point mutations in the gene encoding the leptin receptor, leading to leptin receptor inactivation. Since homozygous db/db mice are sterile, the maintenance of db/db mice requires breeding between heterozygous pairs, which makes genotyping essential for the identification of offspring. The aim of this study was to develop a quick and highly repeatable method for genotyping db/db mice, which comprised only three simple steps: genomic DNA is extracted from either tail tips or ear notches via alkaline lysis (∼20 min); samples are then subjected to tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) using specially designed and validated primer sets (∼1.5 h); finally, genotypes are be determined by resolving PCR products on regular DNA electrophoresis (∼10 min). The entire db/db mice genotyping procedure can be performed using regular Taq polymerase and PCR amplification within 2 h. Other advantages of this method include high sensitivity and reproducibility. Minimal amounts of tissue from mice are required, and genomic DNA samples can be stably stored at room temperature for up to one month. In conclusion, the method is simple, cost effective, sensitive and reliable, which will greatly facilitate studies using db/db mice.

An Essential Regulatory System Originating from Polygenic Transcriptional Rewiring of PhoP-PhoQ of Xanthomonas campestris.

How essential, regulatory genes originate and evolve is intriguing because mutations of these genes not only lead to lethality in organisms, but also have pleiotropic effects since they control the expression of multiple downstream genes. Therefore, the evolution of essential, regulatory genes is not only determined by genetic variations of their own sequences, but also by the biological function of downstream genes and molecular mechanisms of regulation. To understand the origin of essential, regulatory genes, experimental dissection of the complete regulatory cascade is needed. Here, we provide genetic evidences to reveal that PhoP-PhoQ is an essential two-component signal transduction system in the gram-negative bacterium Xanthomonas campestris, but that its orthologs in other bacteria belonging to Proteobacteria are nonessential. Mutational, biochemical, and chromatin immunoprecipitation together with high-throughput sequencing analyses revealed that phoP and phoQ of X. campestris and its close relative Pseudomonas aeruginosa are replaceable, and that the consensus binding motifs of the transcription factor PhoP are also highly conserved. PhoP Xcc in X. campestris regulates the transcription of a number of essential, structural genes by directly binding to cis-regulatory elements (CREs); however, these CREs are lacking in the orthologous essential, structural genes in P. aeruginosa, and thus the regulatory relationships between PhoP Pae and these downstream essential genes are disassociated. Our findings suggested that the recruitment of regulatory proteins by critical structural genes via transcription factor-CRE rewiring is a driving force in the origin and functional divergence of essential, regulatory genes.

Two-Component Signaling System VgrRS Directly Senses Extracytoplasmic and Intracellular Iron to Control Bacterial Adaptation under Iron Depleted Stress.

Both iron starvation and excess are detrimental to cellular life, especially for animal and plant pathogens since they always live in iron-limited environments produced by host immune responses. However, how organisms sense and respond to iron is incompletely understood. Herein, we reveal that in the phytopathogenic bacterium Xanthomonas campestris pv. campestris, VgrS (also named ColS) is a membrane-bound receptor histidine kinase that senses extracytoplasmic iron limitation in the periplasm, while its cognate response regulator, VgrR (ColR), detects intracellular iron excess. Under iron-depleted conditions, dissociation of Fe3+ from the periplasmic sensor region of VgrS activates the VgrS autophosphorylation and subsequent phosphotransfer to VgrR, an OmpR-family transcription factor that regulates bacterial responses to take up iron. VgrR-VgrS regulon and the consensus DNA binding motif of the transcription factor VgrR were dissected by comparative proteomic and ChIP-seq analyses, which revealed that in reacting to iron-depleted environments, VgrR directly or indirectly controls the expressions of hundreds of genes that are involved in various physiological cascades, especially those associated with iron-uptake. Among them, we demonstrated that the phosphorylated VgrR tightly represses the transcription of a special TonB-dependent receptor gene, tdvA. This regulation is a critical prerequisite for efficient iron uptake and bacterial virulence since activation of tdvA transcription is detrimental to these processes. When the intracellular iron accumulates, the VgrR-Fe2+ interaction dissociates not only the binding between VgrR and the tdvA promoter, but also the interaction between VgrR and VgrS. This relieves the repression in tdvA transcription to impede continuous iron uptake and avoids possible toxic effects of excessive iron accumulation. Our results revealed a signaling system that directly senses both extracytoplasmic and intracellular iron to modulate bacterial iron homeostasis.