A concept for international societally relevant microbiology education and microbiology knowledge promulgation in society.

EXECUTIVE SUMMARY: Microbes are all pervasive in their distribution and influence on the functioning and well-being of humans, life in general and the planet. Microbially-based technologies contribute hugely to the supply of important goods and services we depend upon, such as the provision of food, medicines and clean water. They also offer mechanisms and strategies to mitigate and solve a wide range of problems and crises facing humanity at all levels, including those encapsulated in the sustainable development goals (SDGs) formulated by the United Nations. For example, microbial technologies can contribute in multiple ways to decarbonisation and hence confronting global warming, provide sanitation and clean water to the billions of people lacking them, improve soil fertility and hence food production and develop vaccines and other medicines to reduce and in some cases eliminate deadly infections. They are the foundation of biotechnology, an increasingly important and growing business sector and source of employment, and the centre of the bioeconomy, Green Deal, etc. But, because microbes are largely invisible, they are not familiar to most people, so opportunities they offer to effectively prevent and solve problems are often missed by decision-makers, with the negative consequences this entrains. To correct this lack of vital knowledge, the International Microbiology Literacy Initiative-the IMiLI-is recruiting from the global microbiology community and making freely available, teaching resources for a curriculum in societally relevant microbiology that can be used at all levels of learning. Its goal is the development of a society that is literate in relevant microbiology and, as a consequence, able to take full advantage of the potential of microbes and minimise the consequences of their negative activities. In addition to teaching about microbes, almost every lesson discusses the influence they have on sustainability and the SDGs and their ability to solve pressing problems of societal inequalities. The curriculum thus teaches about sustainability, societal needs and global citizenship. The lessons also reveal the impacts microbes and their activities have on our daily lives at the personal, family, community, national and global levels and their relevance for decisions at all levels. And, because effective, evidence-based decisions require not only relevant information but also critical and systems thinking, the resources also teach about these key generic aspects of deliberation. The IMiLI teaching resources are learner-centric, not academic microbiology-centric and deal with the microbiology of everyday issues. These span topics as diverse as owning and caring for a companion animal, the vast range of everyday foods that are produced via microbial processes, impressive geological formations created by microbes, childhood illnesses and how they are managed and how to reduce waste and pollution. They also leverage the exceptional excitement of exploration and discovery that typifies much progress in microbiology to capture the interest, inspire and motivate educators and learners alike. The IMiLI is establishing Regional Centres to translate the teaching resources into regional languages and adapt them to regional cultures, and to promote their use and assist educators employing them. Two of these are now operational. The Regional Centres constitute the interface between resource creators and educators-learners. As such, they will collect and analyse feedback from the end-users and transmit this to the resource creators so that teaching materials can be improved and refined, and new resources added in response to demand: educators and learners will thereby be directly involved in evolution of the teaching resources. The interactions between educators-learners and resource creators mediated by the Regional Centres will establish dynamic and synergistic relationships-a global societally relevant microbiology education ecosystem-in which creators also become learners, teaching resources are optimised and all players/stakeholders are empowered and their motivation increased. The IMiLI concept thus embraces the principle of teaching societally relevant microbiology embedded in the wider context of societal, biosphere and planetary needs, inequalities, the range of crises that confront us and the need for improved decisioning, which should ultimately lead to better citizenship and a humanity that is more sustainable and resilient. ABSTRACT: The biosphere of planet Earth is a microbial world: a vast reactor of countless microbially driven chemical transformations and energy transfers that push and pull many planetary geochemical processes, including the cycling of the elements of life, mitigate or amplify climate change (e.g., Nature Reviews Microbiology, 2019, 17, 569) and impact the well-being and activities of all organisms, including humans. Microbes are both our ancestors and creators of the planetary chemistry that allowed us to evolve (e.g., Life's engines: How microbes made earth habitable, 2023). To understand how the biosphere functions, how humans can influence its development and live more sustainably with the other organisms sharing it, we need to understand the microbes. In a recent editorial (Environmental Microbiology, 2019, 21, 1513), we advocated for improved microbiology literacy in society. Our concept of microbiology literacy is not based on knowledge of the academic subject of microbiology, with its multitude of component topics, plus the growing number of additional topics from other disciplines that become vitally important elements of current microbiology. Rather it is focused on microbial activities that impact us-individuals/communities/nations/the human world-and the biosphere and that are key to reaching informed decisions on a multitude of issues that regularly confront us, ranging from personal issues to crises of global importance. In other words, it is knowledge and understanding essential for adulthood and the transition to it, knowledge and understanding that must be acquired early in life in school. The 2019 Editorial marked the launch of the International Microbiology Literacy Initiative, the IMiLI. HERE, WE PRESENT: our concept of how microbiology literacy may be achieved and the rationale underpinning it; the type of teaching resources being created to realise the concept and the framing of microbial activities treated in these resources in the context of sustainability, societal needs and responsibilities and decision-making; and the key role of Regional Centres that will translate the teaching resources into local languages, adapt them according to local cultural needs, interface with regional educators and develop and serve as hubs of microbiology literacy education networks. The topics featuring in teaching resources are learner-centric and have been selected for their inherent relevance, interest and ability to excite and engage. Importantly, the resources coherently integrate and emphasise the overarching issues of sustainability, stewardship and critical thinking and the pervasive interdependencies of processes. More broadly, the concept emphasises how the multifarious applications of microbial activities can be leveraged to promote human/animal, plant, environmental and planetary health, improve social equity, alleviate humanitarian deficits and causes of conflicts among peoples and increase understanding between peoples (Microbial Biotechnology, 2023, 16(6), 1091-1111). Importantly, although the primary target of the freely available (CC BY-NC 4.0) IMiLI teaching resources is schoolchildren and their educators, they and the teaching philosophy are intended for all ages, abilities and cultural spectra of learners worldwide: in university education, lifelong learning, curiosity-driven, web-based knowledge acquisition and public outreach. The IMiLI teaching resources aim to promote development of a global microbiology education ecosystem that democratises microbiology knowledge.

Microbial biotechnology and beyond: A roadmap for sustainable development and climate mitigation in the transition from fossil fuels to green chemistry.

Our planet, which operates as a closed system, is facing increasing entropy due to human activities such as the overexploitation of natural resources and fossil fuel use. The COP28 in Dubai emphasized the urgency to abandon fossil fuels, recognizing them as the primary cause of human-induced environmental changes, while highlighting the need to transition to renewable energies. We promote the crucial role of microbes for sustaining biogenic cycles to combat climate change and the economic potential of synthetic biology tools for producing diverse non-fossil fuels and chemicals, thus contributing to emission reduction in transport and industry. The shift to 'green chemistry' encounters challenges, derived from the availability of non-food residues and waste (mainly lignocellulosic) as raw material, the construction of cost-effective bioprocessing plants, product recovery from fermentation broths and the utilization of leftover lignin residues for synthesizing new chemicals, aligning with circular economy and sustainable development goals. To meet the Paris Agreement goals, an urgent global shift to low-carbon, renewable sources is imperative, ultimately leading to the cessation of our reliance on fossil fuels.

Weaponising microbes for peace.

There is much human disadvantage and unmet need in the world, including deficits in basic resources and services considered to be human rights, such as drinking water, sanitation and hygiene, healthy nutrition, access to basic healthcare, and a clean environment. Furthermore, there are substantive asymmetries in the distribution of key resources among peoples. These deficits and asymmetries can lead to local and regional crises among peoples competing for limited resources, which, in turn, can become sources of discontent and conflict. Such conflicts have the potential to escalate into regional wars and even lead to global instability. Ergo: in addition to moral and ethical imperatives to level up, to ensure that all peoples have basic resources and services essential for healthy living and to reduce inequalities, all nations have a self-interest to pursue with determination all available avenues to promote peace through reducing sources of conflicts in the world. Microorganisms and pertinent microbial technologies have unique and exceptional abilities to provide, or contribute to the provision of, basic resources and services that are lacking in many parts of the world, and thereby address key deficits that might constitute sources of conflict. However, the deployment of such technologies to this end is seriously underexploited. Here, we highlight some of the key available and emerging technologies that demand greater consideration and exploitation in endeavours to eliminate unnecessary deprivations, enable healthy lives of all and remove preventable grounds for competition over limited resources that can escalate into conflicts in the world. We exhort central actors: microbiologists, funding agencies and philanthropic organisations, politicians worldwide and international governmental and non-governmental organisations, to engage - in full partnership - with all relevant stakeholders, to 'weaponise' microbes and microbial technologies to fight resource deficits and asymmetries, in particular among the most vulnerable populations, and thereby create humanitarian conditions more conducive to harmony and peace.

Insights into the susceptibility of Pseudomonas putida to industrially relevant aromatic hydrocarbons that it can synthesize from sugars.

Pseudomonas putida DOT-T1E is a highly solvent tolerant strain for which many genetic tools have been developed. The strain represents a promising candidate host for the synthesis of aromatic compounds-opening a path towards a green alternative to petrol-derived chemicals. We have engineered this strain to produce phenylalanine, which can then be used as a raw material for the synthesis of styrene via trans-cinnamic acid. To understand the response of this strain to the bioproducts of interest, we have analyzed the in-depth physiological and genetic response of the strain to these compounds. We found that in response to the exposure to the toxic compounds that the strain can produce, the cell launches a multifactorial response to enhance membrane impermeabilization. This process occurs via the activation of a cis to trans isomerase that converts cis unsaturated fatty acids to their corresponding trans isomers. In addition, the bacterial cells initiate a stress response program that involves the synthesis of a number of chaperones and ROS removing enzymes, such as peroxidases and superoxide dismutases. The strain also responds by enhancing the metabolism of glucose through the specific induction of the glucose phosphorylative pathway, Entner-Doudoroff enzymes, Krebs cycle enzymes and Nuo. In step with these changes, the cells induce two efflux pumps to extrude the toxic chemicals. Through analyzing a wide collection of efflux pump mutants, we found that the most relevant pump is TtgGHI, which is controlled by the TtgV regulator.

Rio Tinto as a niche for acidophilus enzymes of industrial relevance.

Lignocellulosic residues are amongst the most abundant waste products on Earth. Therefore, there is an increasing interest in the utilization of these residues for bioethanol production and for biorefineries to produce compounds of industrial interest. Enzymes that breakdown cellulose and hemicellulose into oligomers and monosaccharides are required in these processes and cellulolytic enzymes with optimum activity at a low pH area are desirable for industrial processes. Here, we explore the fungal biodiversity of Rio Tinto, the largest acidic ecosystem on Earth, as far as the secretion of cellulolytic enzymes is concerned. Using colorimetric and industrial substrates, we show that a high proportion of the fungi present in this extremophilic environment secrete a wide range of enzymes that are able to hydrolyze cellulose and hemicellulose at acidic pH (4.5-5). Shotgun proteomic analysis of the secretomes of some of these fungi has identified different cellulases and hemicellulolytic enzymes as well as a number of auxiliary enzymes. Supplementation of pre-industrial cocktails from Myceliophtora with Rio Tinto secretomes increased the amount of monosaccharides released from corn stover or sugar cane straw. We conclude that the Rio Tinto fungi display a good variety of hydrolytic enzymes with high industrial potential.

Synthesis of aromatic amino acids from 2G lignocellulosic substrates.

Pseudomonas putida is a highly solvent-resistant microorganism and useful chassis for the production of value-added compounds from lignocellulosic residues, in particular aromatic compounds that are made from phenylalanine. The use of these agricultural residues requires a two-step treatment to release the components of the polysaccharides of cellulose and hemicellulose as monomeric sugars, the most abundant monomers being glucose and xylose. Pan-genomic studies have shown that Pseudomonas putida metabolizes glucose through three convergent pathways to yield 6-phosphogluconate and subsequently metabolizes it through the Entner-Doudoroff pathway, but the strains do not degrade xylose. The valorization of both sugars is critical from the point of view of economic viability of the process. For this reason, a P. putida strain was endowed with the ability to metabolize xylose via the xylose isomerase pathway, by incorporating heterologous catabolic genes that convert this C5 sugar into intermediates of the pentose phosphate cycle. In addition, the open reading frame T1E_2822, encoding glucose dehydrogenase, was knocked-out to avoid the production of the dead-end product xylonate. We generated a set of DOT-T1E-derived strains that metabolized glucose and xylose simultaneously in culture medium and that reached high cell density with generation times of around 100 min with glucose and around 300 min with xylose. The strains grew in 2G hydrolysates from diluted acid and steam explosion pretreated corn stover and sugarcane straw. During growth, the strains metabolized > 98% of glucose, > 96% xylose and > 85% acetic acid. In 2G hydrolysates P. putida 5PL, a DOT-T1E derivative strain that carries up to five independent mutations to avoid phenylalanine metabolism, accumulated this amino acid in the medium. We constructed P. putida 5PLΔgcd (xylABE) that produced up to 250 mg l-1 of phenylalanine when grown in 2G pretreated corn stover or sugarcane straw. These results support as a proof of concept the potential of P. putida as a chassis for 2G processes.

The soil crisis: the need to treat as a global health problem and the pivotal role of microbes in prophylaxis and therapy.

Soil provides a multitude of services that are essential to a healthily functioning biosphere and continuity of the human race, such as feeding the growing human population and the sequestration of carbon needed to counteract global warming. Healthy soil availability is the limiting parameter in the provision of a number of these services. As a result of anthropogenic abuses, and natural and global warming-promoted extreme weather events, Planet Earth is currently experiencing an unprecedented crisis of soil deterioration, desertification and erosive loss that increasingly prejudices the services it provides. Such services are pivotal to the Sustainability Development Goals formulated by the United Nations. Immediate and coordinated action on a global scale is urgently required to slow and ultimately reverse the loss of healthy soils. Despite the 'dirt-dust', non-vital appearance of soil, it is a highly dynamic living entity, whose life is overwhelmingly microbial. The soil microbiota, which constitutes the greatest reservoir and donor of microbial diversity on Earth, acts as a vast bioreactor, mediating a myriad of chemical reactions that turn the biogeochemical cycles, recycle wastes, purify water, and underpin the multitude of other services soil provides. Fuelling the belowground microbial bioreactor is the aboveground plant and photosynthetic surface microbial life which captures solar energy, fixes inorganic CO2 to organic carbon, and channels fixed carbon and energy into soil. In order to muster an effective response to the crisis, to avoid further deterioration, and to restore unhealthy soils, we need a new and coherent approach, namely to deal with soils worldwide as patients in need of health care and create (i) a public health system for development of effective policies for land use, conservation, restoration, recommendations of prophylactic measures, monitoring and identification of problems (epidemiology), organizing crisis responses, etc., and (ii) a healthcare system charged with soil care: the promotion of good practices, implementation of prophylaxis measures, and institution of therapies for treatment of unhealthy soils and restoration of drylands. These systems need to be national but there is also a desperate need for international coordination. To enable development of effective, evidence-based strategies that will underpin the efforts of soil healthcare systems, a substantial investment in wide-ranging interdisciplinary research on soil health and disease is mandatory. This must lead to a level of understanding of the soil:biota functionalities underlying key ecosystem services that enables formulation of effective diagnosis-prophylaxis-therapy pathways for sustainable use, protection and restoration of different types of soil resources in different climatic zones. These conservation-regenerative-restorative measures need to be complemented by an educative-political-economic-legislative framework that provides incentives encouraging soil care: knowledge, policy, economic and others, and laws which promote international adherence to the principles of restorative soil management. And: we must all be engaged in improving soil health; everyone has a duty of care (https://www.bbc.co.uk/ideas/videos/why-soil-is-one-of-the-most-amazing-things-on-eart/p090cf64). Creative application of microbes, microbiomes and microbial biotechnology will be central to the successful operation of the healthcare systems.

Plant growth-stimulating rhizobacteria capable of producing L-amino acids.

Pseudomonas putida BIRD-1 is a microorganism that inhabits the rhizosphere and solubilizes phosphate and iron and produces indolacetic acid [Roca, A., Pizarro-Tobías, P., Udaondo, Z., Fernández, M., Matilla, M.A., Molina-Henares, M.A., et al. (2013) Analysis of plant growth-promoting properties encoded by the genome of the rhizobacterium Pseudomonas putida BIRD-1. Environ Microbiol 15: 780-794]. In this study, we generated mutant strains that are capable of producing the plant growth stimulating compounds L-tryptophan and L-phenylalanine. We prepared clones that overproduce L-tryptophan by first mutagenizing P. putida BIRD-1, then by selecting for clones in the presence of inhibitory concentrations of 5-fluoro-D,L-tryptophan. The production of this aromatic amino acid was confirmed by chemical analysis and cross-feeding experiments with auxotrophs. One of the mutants, named P. putida BIRD-1-12, was mutagenized again to isolate clones that are also able to grow in the presence of inhibitory concentrations of p-fluoro-D,L-phenylalanine. One of these resulting clones was then isolated and named BIRD-1-12F. Our analysis revealed that the strains that either overproduce L-tryptophan, or L-tryptophan and L-phenylalanine, excel at promoting the growth of a number of plant crops of agricultural interest.

Caring soils for sustainable land uses.

The aim of the article is to develop and explore the idea that soil health is an essential element in combating climate change and promoting food security. An important aspect of this is that soil, rather than simply serving to support plants and as a niche for animals and microbes, also functions as a natural reactor that, through a series of chemical and biological reactions, purifies water, replenish aquifers and maintains equilibria in surface waters. This topic is particularly timely given the recent announcement of the Mission program for Health of Soils by the European Commission. Within this realm, the article intends to catalyze and promote the debate around what defines sustainable agriculture in order to help shape its future.

The versatility of Pseudomonas putida in the rhizosphere environment.

This article addresses the lifestyle of Pseudomonas and focuses on how Pseudomonas putida can be used as a model system for biotechnological processes in agriculture, and in the removal of pollutants from soils. In this chapter we aim to show how a deep analysis using genetic information and experimental tests has helped to reveal insights into the lifestyle of Pseudomonads. Pseudomonas putida is a Plant Growth Promoting Rhizobacteria (PGPR) that establishes commensal relationships with plants. The interaction involves a series of functions encoded by core genes which favor nutrient mobilization, prevention of pathogen development and efficient niche colonization. Certain Pseudomonas putida strains harbor accessory genes that confer specific biodegradative properties and because these microorganisms can thrive on the roots of plants they can be exploited to remove pollutants via rhizoremediation, making the consortium plant/Pseudomonas a useful tool to combat pollution.

Full Transcriptomic Response of Pseudomonas aeruginosa to an Inulin-Derived Fructooligosaccharide.

Pseudomonas aeruginosa is an ubiquitous gram-negative opportunistic human pathogen which is not considered part of the human commensal gut microbiota. However, depletion of the intestinal microbiota (Dysbiosis) following antibiotic treatment facilitates the colonization of the intestinal tract by Multidrug-Resistant P. aeruginosa. One possible strategy is based on the use of functional foods with prebiotic activity. The bifidogenic effect of the prebiotic inulin and its hydrolyzed form (fructooligosaccharide: FOS) is well established since they promote the growth of specific beneficial (probiotic) gut bacteria such as bifidobacteria. Previous studies of the opportunistic nosocomial pathogen Pseudomonas aeruginosa PAO1 have shown that inulin and to a greater extent FOS reduce growth and biofilm formation, which was found to be due to a decrease in motility and exotoxin secretion. However, the transcriptional basis for these phenotypic alterations remains unclear. To address this question we conducted RNA-sequence analysis. Changes in the transcript level induced by inulin and FOS were similar, but a set of transcript levels were increased in response to inulin and reduced in the presence of FOS. In the presence of inulin or FOS, 260 and 217 transcript levels, respectively, were altered compared to the control to which no polysaccharide was added. Importantly, changes in transcript levels of 57 and 83 genes were found to be specific for either inulin or FOS, respectively, indicating that both compounds trigger different changes. Gene pathway analyses of differentially expressed genes (DEG) revealed a specific FOS-mediated reduction in transcript levels of genes that participate in several canonical pathways involved in metabolism and growth, motility, biofilm formation, ß-lactamase resistance, and in the modulation of type III and VI secretion systems; results that have been partially verified by real time quantitative PCR measurements. Moreover, we have identified a genomic island formed by a cluster of 15 genes, encoding uncharacterized proteins, which were repressed in the presence of FOS. The analysis of isogenic mutants has shown that genes of this genomic island encode proteins involved in growth, biofilm formation and motility. These results indicate that FOS selectively modulates bacterial pathogenicity by interfering with different signaling pathways.

High-quality genome-scale metabolic modelling of Pseudomonas putida highlights its broad metabolic capabilities.

Genome-scale reconstructions of metabolism are computational species-specific knowledge bases able to compute systemic metabolic properties. We present a comprehensive and validated reconstruction of the biotechnologically relevant bacterium Pseudomonas putida KT2440 that greatly expands computable predictions of its metabolic states. The reconstruction represents a significant reactome expansion over available reconstructed bacterial metabolic networks. Specifically, iJN1462 (i) incorporates several hundred additional genes and associated reactions resulting in new predictive capabilities, including new nutrients supporting growth; (ii) was validated by in vivo growth screens that included previously untested carbon (48) and nitrogen (41) sources; (iii) yielded gene essentiality predictions showing large accuracy when compared with a knock-out library and Bar-seq data; and (iv) allowed mapping of its network to 82 P. putida sequenced strains revealing functional core that reflect the large metabolic versatility of this species, including aromatic compounds derived from lignin. Thus, this study provides a thoroughly updated metabolic reconstruction and new computable phenotypes for P. putida, which can be leveraged as a first step toward understanding the pan metabolic capabilities of Pseudomonas.

Regulation of carbohydrate degradation pathways in Pseudomonas involves a versatile set of transcriptional regulators.

Bacteria of the genus Pseudomonas are widespread in nature. In the last decades, members of this genus, especially Pseudomonas aeruginosa and Pseudomonas putida, have acquired great interest because of their interactions with higher organisms. Pseudomonas aeruginosa is an opportunistic pathogen that colonizes the lung of cystic fibrosis patients, while P. putida is a soil bacterium able to establish a positive interaction with the plant rhizosphere. Members of Pseudomonas genus have a robust metabolism for amino acids and organic acids as well as aromatic compounds; however, these microbes metabolize a very limited number of sugars. Interestingly, they have three-pronged metabolic system to generate 6-phosphogluconate from glucose suggesting an adaptation to efficiently consume this sugar. This review focuses on the description of the regulatory network of glucose utilization in Pseudomonas, highlighting the differences between P. putida and P. aeruginosa. Most interestingly, It is highlighted a functional link between glucose assimilation and exotoxin A production in P. aeruginosa. The physiological relevance of this connection remains unclear, and it needs to be established whether a similar relationship is also found in other bacteria.

Ruminal metagenomic libraries as a source of relevant hemicellulolytic enzymes for biofuel production.

The success of second-generation (2G) ethanol technology relies on the efficient transformation of hemicellulose into monosaccharides and, particularly, on the full conversion of xylans into xylose for over 18% of fermentable sugars. We sought new hemicellulases using ruminal liquid, after enrichment of microbes with industrial lignocellulosic substrates and preparation of metagenomic libraries. Among 150 000 fosmid clones tested, we identified 22 clones with endoxylanase activity and 125 with ß-xylosidase activity. These positive clones were sequenced en masse, and the analysis revealed open reading frames with a low degree of similarity with known glycosyl hydrolases families. Among them, we searched for enzymes that were thermostable (activity at > 50°C) and that operate at high rate at pH around 5. Upon a wide series of assays, the clones exhibiting the highest endoxylanase and ß-xylosidase activities were identified. The fosmids were sequenced, and the corresponding genes cloned, expressed and proteins purified. We found that the activity of the most active ß-xylosidase was at least 10-fold higher than that in commercial enzymatic fungal cocktails. Endoxylanase activity was in the range of fungal enzymes. Fungal enzymatic cocktails supplemented with the bacterial hemicellulases exhibited enhanced release of sugars from pretreated sugar cane straw, a relevant agricultural residue.

The contribution of microbial biotechnology to economic growth and employment creation.

Our communication discusses the profound impact of bio-based economies - in particular microbial biotechnologies - on SDG 8: Promote sustained, inclusive and sustainable economic growth, full and productive employment and decent work for all. A bio-based economy provides significant potential for improving labour supply, education and investment, and thereby for substantially increasing the demographic dividend. This, in turn, improves the sustainable development of economies.

The contribution of microbial biotechnology to sustainable development goals.

The signature and almost unique characteristic of microbial technology is the exceptional diversity of applications it can address, and the exceptional range of human activities and needs to which it is and can be applied. Precisely because sustainability goals have very diverse and complex components and requirements, microbial technology has the ability to contribute substantively on many levels in many arenas to global efforts to achieve sustainability. Indeed, microbial technology could be viewed as a unifying element in our progress towards sustainability.

Identification and elucidation of in vivo function of two alanine racemases from Pseudomonas putida KT2440.

The genome of Pseudomonas putida KT2440 contains two open reading frames (ORFs), PP_3722 and PP_5269, that encode proteins with a Pyridoxal phosphate binding motif and a high similarity to alanine racemases. Alanine racemases play a key role in the biosynthesis of D-alanine, a crucial amino acid in the peptidoglycan layer. For these ORFs, we generated single and double mutants and found that inactivation of PP_5269 resulted in D-alanine auxotrophy, while inactivation of PP_3722 did not. Furthermore, as expected, the PP_3722/PP_5269 double mutant was a strict auxotroph for D-alanine. These results indicate that PP_5269 is an alr allele and that it is the essential alanine racemase in P. putida. We observed that the PP_5269 mutant grew very slowly, while the double PP_5269/PP_3722 mutant did not grow at all. This suggests that PP_3722 may replace PP_5269 in vivo. In fact, when the ORF encoding PP_3772 was cloned into a wide host range expression vector, ORF PP_3722 successfully complemented P. putida PP_5269 mutants. We purified both proteins to homogeneity and while they exhibit similar KM values, the Vmax of PP_5269 is fourfold higher than that of PP_3722. Here, we propose that PP_5269 and PP_3722 encode functional alanine racemases and that these genes be named alr-1 and alr-2 respectively.