Chemical and biological understanding of fluorine in its many forms has lagged behind that of the other halogens, but studies on fluorine have recently come to the forefront. Thousands of commercial fluorine-containing materials are now found throughout the fabric of society, prompting new concerns regarding their health and environmental impacts. The utility and fate of many fluorine-containing products are inextricably linked to microbial responses to the element in its various forms. In this issue, the paper by Calero et al. (2022) provides new insights into the response of the model soil bacterium Pseudomonas putida KT2440 to fluoride anion. The study takes a broad approach to paint a picture of how fluoride impacts the bacterium and the microorganism's physiological reaction. Methods employed include fluoride toxicity assays, Tn-Seq, genetic knockouts, fluorescent sensing of fluoride intracellularly and metabolomics. Clearly, microbes like P. putida KT2440 have evolved multiple mechanisms to protect themselves from toxic fluoride anion.

Fluorine is the 13th most abundant element in the Earth's crust but the unavailability of mineral forms and its cellular toxicity has limited the element in bacteria. A review article compiling information on the 33 most abundant elements in prokaryotes did not detect fluorine, showing it to be less prevalent than cadmium, tin and barium in the organisms examined (Novoselov et al., 2013). However, a select few bacteria and plants have learned to sequester fluoride for the purpose of biosynthesizing monofluorinated anti-metabolites as toxins that ward off predators (Chan & O'Hagan, 2012; Walker & Chang, 2014). There is currently interest in producing new fluorinated compounds using the enzyme fluorinase (Calero et al., 2020; O'Hagan & Deng, 2015; Pardo et al., 2022) and this is one of the potential outgrowths of the present work by Calero et al.

The scarcity of naturally biosynthesized fluorinated compounds by microbes and plants likely emanates at least partly from cellular toxicity of the mineral acid hydrogen fluoride, HF, and its dissociated anion, fluoride. Humans tragically experienced the toxicity of fluorine gas (F₂) and HF in 19th-century laboratories (Weeks, 1932). Henri Moissan was honoured with the Nobel Prize in Chemistry in 1906 for his innovations in safer handling of fluorine but unfortunately died several months after receipt of the award. Subsequent to Moissan, there was a great expansion in industrial uses of fluorine, leading to Freon refrigerants, Teflon-type polymers, specialized surfactants, and most recently, agrichemicals and pharmaceuticals (Dolbier Jr, 2005; Lombardo, 1981). Unlike F₂ and HF, many commercial organofluorine compounds are largely unreactive with microbial enzymes, leading to an undesirable environmental persistence (Wackett, 2021).

Most industrial fluorine today derives from the mineral fluorite (CaF₂). CaF₂ is converted to HF and salts of the conjugate base, fluoride anion. Fluoride is used in the types of organofluorine synthesis previously mentioned, as well as aluminium extraction, steel hardening and water fluoridation (Pelham, 1985). Related to the latter, fluoride is added to toothpastes to harden teeth and inhibit caries-causing oral bacteria such as Streptococcus mutans (Marquis, 1995). The application of inhibiting microbes points to the major differences in microbiological response to fluoride compared to chloride anion. Chloride anion is abundant in many bacterial cells at concentrations greater than 50 mM and some halophiles prefer molar levels of the anion (Chen, 2005). By contrast, fluoride anion shows toxicity at intracellular levels of 0.1 mM (Ji et al., 2014; McIlwain et al., 2021). At that level and above, fluoride coordinates to metal centres in key enzymes, such as ATPases, pyrophosphatase and enolase. Fluoride stress is increased by low pH as HF is the species that enters cells, which then dissociates to fluoride anion at the near-neutral pH values of the cytoplasm in most bacteria (Ji et al., 2014).

To respond, cells must initially sense potentially toxic levels of fluoride intracellularly and that can be mediated, in some cases, by a fluoride-responsive riboswitch which controls gene expression (Baker et al., 2012; Breaker, 2012). Among the most important genes are those encoding membrane proteins that transport fluoride out of the cytoplasm (Ji et al., 2014; Last et al., 2018; Stockbridge et al., 2012). One such protein is a fluoride-proton antiporter and another is a fluoride exporter in which fluoride expulsion is driven by an electrochemical gradient. Indeed, a major gene upregulated in Pseudomonas putida KT2440 during fluoride exposure, as shown in the paper by Calero et al. (2022), is crcB that encodes the gradient-driven fluoride exporter. The X-ray structure of the CrcB protein from Bordetella pertussis has been solved to a maximum resolution of 2.1 Å (Stockbridge et al., 2015) and CrcB transporters have been shown to export fluoride anion at an astounding rate of 10⁵ per second (McIlwain et al., 2021).

Basic and directed fluoride response work has previously been conducted with E. coli and Streptococcus spp., respectively, the latter due to the cavity-prevention angle (Ji et al., 2014; Liao et al., 2017). The current study by Calero et al. (2022) is important in the context of it focusing on a model soil bacterium, P. putida KT2440 (Belda et al., 2016), and using multiple molecular genetic approaches to obtain new insights into responses to fluoride. The article describes the use of a Tn-seq library and making scar-less deletion mutants of fluoride-responsive genes thus identified. The authors constructed an intracellular fluoride biosensor to determine internal fluoride levels under different environmental conditions with multiple mutants. The study also explores pH and metabolic perturbations occurring with fluoride insult. Previous work had shown fluoride sensitivity increases with lower pH (Ji et al., 2014) suggesting that metabolic rerouting, as described in Calero et al. (2022), might raise pH in the cell's local environment and thus mitigate against HF import.

Pseudomonas putida is naturally quite resistant to fluoride and a deeper understanding of its metabolism and genetics is emerging. In this context, P. putida is a good platform for producing novel fluorinated compounds via the fluorinase pathway, which will require high levels of fluoride in the medium, which might be damaging to host organisms lacking stout defence mechanisms (Calero et al., 2020). Moreover, Pseudomonas spp. are recognized as important for biodegradation and may help remediate polyfluorinated pollutants, which will release fluoride from C-F bond cleavage reactions (Wackett, 2022). There are some indicators from environmental studies that Pseudomonas spp. may be prevalent community members in fluoride-laden waters (Zhang et al., 2019).

As all good studies do, the paper by Calero et al. (2022) opens as many new questions as it answers. The identification of genes that respond to fluoride begs new questions regarding their physiological function. The response and importance of the CrcB protein were underscored in the present study. But some upregulated genes are denoted as hypotheticals and even for ‘known’ proteins, the annotated function appears disconnected from fluoride resistance. Indeed, the CrcB protein, which clearly functions as a fluoride exporter, was once designated as a ‘camphor resistance protein’ in E. coli (Hu et al., 1996). This illustrates a fundamental issue in genome annotation, many gene function designations are misleading, some are verifiably wrong (Schnoes et al., 2009). But the paper by Calero et al. (2022) provides the fodder for new discovery, and more precision in annotation related to bacterial response to fluorine, exactly the process by which the science will advance.

对多种形式的氟的化学和生物学理解落后于对其他卤素的理解，但最近对氟的研究走到了前沿。现在在整个社会结构中发现了数以千计的商用含氟材料，这引发了人们对其健康和环境影响的新担忧。许多含氟产品的效用和命运与微生物对各种形式元素的反应密不可分。在本期中，Calero 等人的论文。( 2022 ) 提供了对模型土壤细菌恶臭假单胞菌反应的新见解KT2440 为氟阴离子。该研究采用广泛的方法来描绘氟化物如何影响细菌和微生物的生理反应。采用的方法包括氟化物毒性测定、Tn -Seq、基因敲除、细胞内氟化物的荧光传感和代谢组学。显然，像恶臭假单胞菌KT2440 这样的微生物已经进化出多种机制来保护自己免受有毒氟化物阴离子的侵害。

氟是地壳中第 13 位最丰富的元素，但矿物形式的不可用性及其细胞毒性限制了细菌中的元素。一篇汇编有关原核生物中 33 种最丰富元素的信息的评论文章没有检测到氟，表明它在所检查的生物体中的普遍性低于镉、锡和钡（Novoselov 等人，  2013 年）。然而，少数细菌和植物已经学会隔离氟化物，以生物合成单氟化抗代谢物作为抵御捕食者的毒素（Chan & O'Hagan，  2012 年；Walker & Chang，  2014 年）。目前有兴趣使用氟化酶生产新的氟化化合物（Calero 等人，  2020 年）; 奥哈根和邓，  2015 年；Pardo 等人，  2022 年），这是 Calero 等人目前工作的潜在成果之一。

微生物和植物天然生物合成的氟化化合物的稀缺性可能至少部分源于无机酸氟化氢 HF 及其游离阴离子氟化物的细胞毒性。人类在 19 世纪的实验室中 悲惨地经历了氟气 (F _{2 ) 和 HF 的毒性（Weeks，} 1932 年）。Henri Moissan 因其在更安全地处理氟方面的创新而于 1906 年荣获诺贝尔化学奖，但不幸的是，他在获奖几个月后就去世了。在 Moissan 之后，氟的工业用途有了很大的扩展，导致了氟利昂制冷剂、聚四氟乙烯类聚合物、专用表面活性剂，以及最近的农业化学品和药物（Dolbier Jr，  2005 年；Lombardo， 1981 年）。与 F ₂和 HF 不同，许多商用有机氟化合物在很大程度上不与微生物酶发生反应，从而导致不良的环境持久性（Wackett，  2021 年）。

今天大多数工业氟都来自矿物萤石 (CaF ₂ )。CaF ₂转化为HF和共轭碱、氟阴离子的盐。氟化物用于前面提到的有机氟合成类型，以及铝提取、钢硬化和水氟化 (Pelham,  1985 )。与后者相关的是，氟化物被添加到牙膏中以硬化牙齿并抑制引起龋齿的口腔细菌，例如变形链球菌（Marquis，  1995 年）). 抑制微生物的应用表明，与氯阴离子相比，微生物对氟化物的反应存在主要差异。氯离子在许多细菌细胞中含量丰富，浓度大于 50 mM，一些嗜盐菌更喜欢摩尔水平的阴离子 (Chen,  2005 )。相比之下，氟阴离子在细胞内水平为 0.1 mM 时显示出毒性（Ji 等人，  2014 年；McIlwain 等人，  2021 年）。在该水平及以上水平，氟化物与关键酶（例如 ATP 酶、焦磷酸酶和烯醇酶）中的金属中心配位。低 pH 值会增加氟化物压力，因为 HF 是进入细胞的物质，然后在大多数细菌的细胞质接近中性 pH 值时解离成氟化物阴离子 (Ji et al.,  2014 )。

为了做出反应，细胞必须首先感知细胞内氟化物的潜在毒性水平，并且在某些情况下，可以通过控制基因表达的氟化物反应性核糖开关进行介导（Baker 等人，  2012 年；Breaker，  2012 年）。其中最重要的基因是那些编码将氟化物转运出细胞质的膜蛋白的基因（Ji 等人，  2014 年；Last 等人，  2018 年；Stockbridge 等人，  2012 年）。一种这样的蛋白质是氟质子逆向转运蛋白，另一种是氟化物输出蛋白，其中氟化物排出由电化学梯度驱动。事实上，恶臭假单胞菌中的一个主要基因上调KT2440 在氟化物暴露期间，如 Calero 等人的论文所示。( 2022 )，是编码梯度驱动的氟化物输出器的crcB 。来自百日咳博德特氏菌的 CrcB 蛋白的 X 射线结构已被解析到最大分辨率为 2.1 Å（Stockbridge 等人，  2015 年），并且 CrcB 转运蛋白已被证明以每秒10 ⁵个惊人的速度输出氟化物阴离子（ McIlwain 等人，  2021 年）。

之前已经分别对大肠杆菌和链球菌进行了基本和定向氟化物反应工作，后者由于空腔预防角度（Ji 等人，  2014 年；Liao 等人，  2017 年）。Calero 等人目前的研究。( 2022 ) 在关注模型土壤细菌恶臭假单胞菌KT2440（Belda 等人，  2016 年）并使用多种分子遗传学方法获得对氟化物反应的新见解的背景下很重要。文章介绍了使用Tn- seq文库并制作氟化物反应基因的无疤痕缺失突变体。作者构建了一个细胞内氟化物生物传感器，用于测定不同环境条件下多个突变体的内部氟化物水平。该研究还探讨了因氟化物损害而发生的 pH 值和代谢扰动。之前的工作表明，氟化物敏感性随着 pH 值的降低而增加（Ji 等人，  2014 年），这表明代谢重新路由，如 Calero 等人所述。( 2022 ), 可能会提高细胞局部环境的 pH 值，从而减轻 HF 的输入。

恶臭假单胞菌天然对氟化物具有很强的抵抗力，对其代谢和遗传学的深入了解正在浮现。在这种情况下，恶臭假单胞菌是通过氟化酶途径生产新型氟化化合物的良好平台，这将需要培养基中含有高浓度的氟化物，这可能对缺乏坚固防御机制的宿主生物造成损害（Calero 等人，  2020 年） ). 此外，假单胞菌属。被认为对生物降解很重要，可能有助于修复多氟化污染物，多氟化物会从 CF 键断裂反应中释放氟化物（Wackett，  2022 年）。环境研究中有一些指标表明，假单胞菌属 可能是富含氟化物的水域中普遍存在的社区成员（Zhang 等人，  2019 年）。

正如所有好的研究所做的那样，Calero 等人的论文。( 2022 ) 提出的新问题和它回答的一样多。对氟化物有反应的基因的鉴定引出了有关其生理功能的新问题。本研究强调了 CrcB 蛋白的反应和重要性。但一些上调基因被表示为假设性基因，甚至对于“已知”蛋白质，注释功能似乎与氟化物抗性无关。事实上，CrcB 蛋白，其明显作为氟化物输出蛋白发挥作用，曾被指定为大肠杆菌中的“樟脑抗性蛋白” （Hu 等人，  1996）。这说明了基因组注释中的一个基本问题，许多基因功能名称具有误导性，有些是可验证的错误（Schnoes 等人， 2009 年）。但是 Calero 等人的论文。( 2022 ) 为新发现提供了素材，并更精确地注释了与细菌对氟的反应相关的信息，这正是科学进步的过程。