Engineering of Shewanella marisflavi BBL25 for biomass-based polyhydroxybutyrate production and evaluation of its performance in electricity production.

Polyhydroxybutyrate (PHB) is a biodegradable plastic with physical properties similar to petrochemically derived plastics. Here, Shewanella marisflavi BBL25 was engineered by inserting the pLW487 vector containing polyhydroxyalkanoates synthesis genes from Ralstonia eutropha H16. Under optimal conditions, the engineered S. marisflavi BBL25 produced 1.99 ± 0.05 g/L PHB from galactose. The strain showed high tolerance to various inhibitors and could utilize lignocellulosic biomass for PHB production. When barley straw hydrolysates were used as a carbon source, PHB production was 3.27 ± 0.19 g/L. In addition, PHB production under the microbial fuel cell system was performed to confirm electricity coproduction. The maximum electricity current output density was 1.71 mA/cm2, and dry cell weight (DCW) and PHB production were 11.4 g/L and 6.31 g/L, respectively. Our results demonstrated PHB production using various lignocellulosic biomass and the feasibility of PHB and electricity production, simultaneously, and it is the first example of PHB production in engineered Shewanella.

Survival of the first rather than the fittest in a Shewanella electrode biofilm.

For natural selection to operate there must exist heritable variation among individuals that affects their survival and reproduction. Among free-living microbes, where differences in growth rates largely define selection intensities, competitive exclusion is common. However, among surface attached communities, these dynamics become less predictable. If extreme circumstances were to dictate that a surface population is immortal and all offspring must emigrate, the offspring would be unable to contribute to the composition of the population. Meanwhile, the immortals, regardless of reproductive capacity, would remain unchanged in relative abundance. The normal cycle of birth, death, and competitive exclusion would be broken. We tested whether conditions required to set up this idealized scenario can be approximated in a microbial biofilm. Using two differentially-reproducing strains of Shewanella oneidensis grown on an anode as the sole terminal electron acceptor - a system in which metabolism is obligately tied to surface attachment - we found that selection against a slow-growing competitor is drastically reduced. This work furthers understanding of natural selection dynamics in sessile microbial communities, and provides a framework for designing stable microbial communities for industrial and experimental applications.

Iron can be microbially extracted from Lunar and Martian regolith simulants and 3D printed into tough structural materials.

In-situ resource utilization (ISRU) is increasingly acknowledged as an essential requirement for the construction of sustainable extra-terrestrial colonies. Even with decreasing launch costs, the ultimate goal of establishing colonies must be the usage of resources found at the destination of interest. Typical approaches towards ISRU are often constrained by the mass and energy requirements of transporting processing machineries, such as rovers and massive reactors, and the vast amount of consumables needed. Application of self-reproducing bacteria for the extraction of resources is a promising approach to reduce these pitfalls. In this work, the bacterium Shewanella oneidensis was used to reduce three different types of Lunar and Martian regolith simulants, allowing for the magnetic extraction of iron-rich materials. The combination of bacterial treatment and magnetic extraction resulted in a 5.8-times higher quantity of iron and 43.6% higher iron concentration compared to solely magnetic extraction. The materials were 3D printed into cylinders and the mechanical properties were tested, resulting in a 400% improvement in compressive strength in the bacterially treated samples. This work demonstrates a proof of concept for the on-demand production of construction and replacement parts in space exploration.

Chitosan-riboflavin composite film based on photodynamic inactivation technology for antibacterial food packaging.

Photodynamic inactivation (PDI) is a novel sterilization technology that has proven effective in medicine. This study focused on applying PDI to food packaging, where chitosan (CS) films containing photosensitizing riboflavin (RB) were prepared via solution casting. The CS-RB composite films exhibited good ultraviolet (UV)-barrier properties, and had a visually appealing highly transparent yellow appearance. Scanning electron microscopy (SEM) confirmed even dispersion of RB throughout the CS film. The addition of RB led to improved film characteristics, including the thickness, mechanical properties, solubility, and water barrier properties. The CS-RB5 composite films produced sufficient singlet oxygen under blue LED irradiation for 2 h to inactivate two food-borne pathogens (Listeria monocytogenes and Vibrio parahaemolyticus) and one spoilage bacteria (Shewanella baltica). The CS-RB composite films were assessed as a salmon packaging material, where inhibition of bacterial growth was observed. The film is biodegradable, and has the potential to alleviate the issues associated with the excessive use of petrochemical materials, such as environmental pollution and limited resources. The CS-RB composite films showed potential as a novel environmentally friendly packaging material for shelf-life extension of refrigerated food products.

Modulation of the RNA polymerase activity by AtcB, a protein associated with a DnaK chaperone network in Shewanella oneidensis.

Bacteria possess several molecular pathways to adapt to changing environments and to stress conditions. One of these pathways involves a complex network of chaperone proteins that together control proteostasis. In the aquatic bacterium Shewanella oneidensis, we have recently identified a previously unknown co-chaperone of the DnaK/Hsp70 chaperone system, AtcJ, that is essential for adaptation to low temperatures. AtcJ is encoded in the atcJABC operon, whose products, together with DnaK, form a protein network allowing growth at low temperature. However, how these proteins allow cold adaptation is unknown. Here, we found that AtcB directly interacts with the RNA polymerase and decreases its activity. In addition, AtcB overproduction prevents bacterial growth due to RNA polymerase inhibition. Together, these results suggest that the Atc proteins could direct the DnaK chaperone to the RNA polymerase to sustain life at low temperatures.

Nanoliter scale electrochemistry of natural and engineered electroactive bacteria.

Bacterial extracellular electron transfer (EET) is envisioned for use in applied biotechnologies, necessitating electrochemical characterization of natural and engineered electroactive biofilms under conditions similar to the target application, including small-scale biosensing or biosynthesis platforms, which is often distinct from standard 100 mL-scale stirred-batch bioelectrochemical test platforms used in the laboratory. Here, we adapted an eight chamber, nanoliter volume (500 nL) electrochemical flow cell to grow biofilms of both natural (Biocathode MCL community, Marinobacter atlanticus, and Shewanella oneidensis MR1) or genetically modified (S. oneidensis ΔMtr and S. oneidensis ΔMtr + pLB2) electroactive bacteria on electrodes held at a constant potential. Maximum current density achieved by unmodified strains was similar between the nano- and milliliter-scale reactors. However, S. oneidensis biofilms engineered to activate EET upon exposure to 2,4-diacetylphloroglucinol (DAPG) produced current at wild-type levels in the stirred-batch reactor, but not in the nanoliter flow cell. We hypothesize this was due to differences in mass transport of DAPG, naturally-produced soluble redox mediators, and oxygen between the two reactor types. Results presented here demonstrate, for the first time, nanoliter scale chronoamperometry and cyclic voltammetry of a range of electroactive bacteria in a three-electrode reactor system towards development of miniaturized, and potentially high throughput, bioelectrochemical platforms.

Adaptation of the Marine Bacterium Shewanella baltica to Low Temperature Stress.

Marine bacteria display significant versatility in adaptation to variations in the environment and stress conditions, including temperature shifts. Shewanella baltica plays a major role in denitrification and bioremediation in the marine environment, but is also identified to be responsible for spoilage of ice-stored seafood. We aimed to characterize transcriptional response of S. baltica to cold stress in order to achieve a better insight into mechanisms governing its adaptation. We exposed bacterial cells to 8 °C for 90 and 180 min, and assessed changes in the bacterial transcriptome with RNA sequencing validated with the RT-qPCR method. We found that S. baltica general response to cold stress is associated with massive downregulation of gene expression, which covered about 70% of differentially expressed genes. Enrichment analysis revealed upregulation of only few pathways, including aminoacyl-tRNA biosynthesis, sulfur metabolism and the flagellar assembly process. Downregulation was observed for fatty acid degradation, amino acid metabolism and a bacterial secretion system. We found that the entire type II secretion system was transcriptionally shut down at low temperatures. We also observed transcriptional reprogramming through the induction of RpoE and repression of RpoD sigma factors to mediate the cold stress response. Our study revealed how diverse and complex the cold stress response in S. baltica is.

Aerobic radical polymerization mediated by microbial metabolism.

Performing radical polymerizations under ambient conditions is a major challenge because molecular oxygen is an effective radical quencher. Here we show that the facultative electrogen Shewanella oneidensis can control metal-catalysed living radical polymerizations under apparent aerobic conditions by first consuming dissolved oxygen via aerobic respiration, and then directing extracellular electron flux to a metal catalyst. In both open and closed containers, S. oneidensis enabled living radical polymerizations without requiring the preremoval of oxygen. Polymerization activity was closely tied to S. oneidensis anaerobic metabolism through specific extracellular electron transfer proteins and was effective for a variety of monomers using low (parts per million) concentrations of metal catalysts. Finally, polymerizations survived repeated challenges of oxygen exposure and could be initiated using lyophilized or spent (recycled) cells. Overall, our results demonstrate how the unique ability of S. oneidensis to use both oxygen and metals as respiratory electron acceptors can be leveraged to address salient challenges in polymer synthesis.

Flagellation of Shewanella oneidensis Impacts Bacterial Fitness in Different Environments.

Flagella occur on many prokaryotes, which primarily propel cells to move from detrimental to favorable environments. A variety of species-specific flagellation patterns have been identified. Although it is presumed that for each of these flagellated microorganisms, an evolutionarily fixed flagellation pattern is favored under the normal living conditions, direct evidence is lacking. Here, we use Shewanella oneidensis, a rod-shaped Gram-negative bacterium with a monotrichous polar flagellum (MR-1, the wild-type), as a research model. The investigation has been enabled by multiple mutants with diverse flagellation patterns that had been generated by removing FlhF and FlhG proteins that control flagellar location and number, respectively. Growth assays, as a measure of fitness, revealed that the wild-type strain predominated in spreading on swim plates and in pellicles which form at the air-liquid interface. However, under the pellicles where oxygen is limited, both aflagellated and monotrichous lateral strains showed similar increase in fitness, whereas strains with multiple flagella were less competitive. Moreover, under shaking culturing conditions, the aflagellated strain outcompeted all other strains, including the wild-type, suggesting that cells devoid of flagella would be more likely enriched upon agitation. Overall, these data support the presumption that the monotrichous polar flagellum, as evolutionarily fixed in the wild-type strain, is optimal for the growth fitness of S. oneidensis over any other mutants under most test conditions. However, upon specific changes of environmental conditions, another form could come to predominate. These findings provide insight into the impacts of flagellation patterns and function on bacterial adaptation to differing environments.

Cultivation of Exoelectrogenic Bacteria in Conductive DNA Nanocomposite Hydrogels Yields a Programmable Biohybrid Materials System.

The use of living microorganisms integrated within electrochemical devices is an expanding field of research, with applications in microbial fuel cells, microbial biosensors or bioreactors. We describe the use of porous nanocomposite materials prepared by DNA polymerization of carbon nanotubes (CNTs) and silica nanoparticles (SiNPs) for the construction of a programmable biohybrid system containing the exoelectrogenic bacterium Shewanella oneidensis. We initially demonstrate the electrical conductivity of the CNT-containing DNA composite by employment of chronopotentiometry, electrochemical impedance spectroscopy, and cyclic voltammetry. Cultivation of Shewanella oneidensis in the conductive materials shows that the exoelectrogenic bacteria populate the matrix of the conductive composite, while nonexoelectrogenic Escherichia coli remain on its surface. Moreover, the ability to use extracellular electron transfer pathways is positively correlated with the number of cells within the conductive synthetic biofilm matrix. The Shewanella-containing composite remains stable for several days and shows electrochemical activity, indicating that the conductive backbone is capable of extracting the metabolic electrons produced by the bacteria under strictly anoxic conditions and conducting them to the anode. Programmability of this biohybrid material system is demonstrated by on-demand release and degradation induced by a short-term enzymatic stimulus. We believe that the application possibilities of such biohybrid materials could even go beyond microbial biosensors, bioreactors, and fuel cell systems.

Deteriorated biofilm-forming capacity and electroactivity of Shewanella oneidnsis MR-1 induced by insertion sequence (IS) elements.

Shewanella oneidensis MR-1, a model species of exoelectrogenic bacteria (EEB), has been widely applied in bioelectrochemical systems. Biofilms of EEB grown on electrodes are essential in governing the current output and power density of bioelectrochemical systems. The MR-1 genome is exceptionally dynamic due to the existence of a large number of insertion sequence (IS) elements. However, to date, the impacts of IS elements on the biofilm-forming capacity of EEB and performance of bioelectrochemical systems remain unrevealed. Herein, we isolated a non-motile mutant (NMM) with biofilm-deficient phenotype from MR-1. We found that the insertion of an ISSod2 element into the flrA (encoding the master regulator for flagella synthesis and assembly) of MR-1 resulted in the non-motile and biofilm-deficient phenotypes in NMM cells. Notably, such a variant was readily confused with the wild-type strain because there were no obvious differences in growth rates and colonial morphologies between the two strains. However, the reduced biofilm formation on the electrodes and the deteriorated performances of bioelectrochemical systems and Cr(VI) immobilization for the strain NMM were observed. Given the wide distribution of IS elements in EEB, appropriate cultivation and preservation conditions should be adopted to reduce the likelihood that IS elements-mediated mutation occurs in EEB. These findings reveal the negative impacts of IS elements on the biofilm-forming capacity of EEB and performance of bioelectrochemical systems and suggest that great attention should be given to the actual physiological states of EEB before their applications.

Accelerating itaconic acid production by increasing membrane permeability of whole-cell biocatalyst based on a psychrophilic bacterium Shewanella livingstonensis Ac10.

Whole-cell biocatalysts have numerous advantages including ease of preparation and coenzyme recovery over purified industrially used enzymes. However, the cell membrane can occasionally hinder cytoplasmic diffusion of the substrate, resulting in reduced biotransformation efficiency. Psychrophiles can grow and reproduce at low temperatures; their cell membranes are highly flexible, and their permeability can be improved via heat treatment at a moderate temperature. The aim of this study was to generate a psychrophile-based simple biocatalyst (PSCats) using Shewanella livingstonensis Ac10. This biocatalyst contained two enzymes that were heterologously expressed and converted citric acid to itaconic acid, thereby serving as a potential platform replacing the petroleum-based counterparts. The efficiency of the biocatalyst was increased via heat treatment at 45 °C for 15 min, and itaconic acid productivity of the cells after heat treatment (1.41 g/L/h) was increased around 6-fold in comparison with those without heat treatment (0.22 g/L/h). A large part of the productivity remained (67.3 %) when the cells were reused for 5 times (10 h for each reaction). Therefore, the potential of this heat-permeabilized psychrophile host to increase the productivity of whole-cell biocatalyst was proved; however, further research is necessary to understand the underlying mechanism.

Development of a RP-HPLC method for determination of glucose in Shewanella oneidensis cultures utilizing 1-phenyl-3-methyl-5-pyrazolone derivatization.

A method was developed and validated for low-level detection of glucose. The method involves quantitation of glucose though derivitization with 1-phenyl-3-methyl-5-pyrazolone (PMP) and HPLC-DAD analysis. The developed method was found to be accurate and robust achieving detection limits as low as 0.09 nM. The applicability of the method was tested against microbial samples with glucose acting as a carbon fuel source. The method was shown to be able to accurately discriminate and quantify PMP-glucose derivatives within Shewanella oneidensis MR-1 samples. The method proved capable at examining glucose usage during the early hours of microbial growth, with detectable usage occurring as early as two hours. S. oneidensis cultures were found to grow more effectively in the presence of oxygen which coincided with more efficient glucose usage. Glucose usage further increased in the presence of competing electron acceptors. The rate at which S. oneidensis reached exponential growth was affected by the presence of ferric iron under microaerobic conditions. Such samples reached exponential growth approximately two hours sooner than aerobic samples.

Cobalt Release from a Nanoscale Multiphase Lithiated Cobalt Phosphate Dominates Interaction with Shewanella oneidensis MR-1 and Bacillus subtilis SB491.

Cobalt phosphate engineered nanomaterials (ENMs) are an important class of materials that are used as lithium ion battery cathodes, catalysts, and potentially as super capacitors. As production of these nanomaterials increases, so does the likelihood of their environmental release; however, to date, there are relatively few investigations of the impact of nanoscale metal phosphates on biological systems. Furthermore, nanomaterials used in commercial applications are often multiphase materials, and analysis of the toxic potential of mixtures of nanomaterials has been rare. In this work, we studied the interactions of two model environmental bacteria, Shewanella oneidensis MR-1 and Bacillus subtilis, with a multiphase lithiated cobalt phosphate (mLCP) nanomaterial. Using a growth-based viability assay, we found that mLCP was toxic to both bacteria used in this study. To understand the observed toxicity, we screened for production of reactive oxygen species (ROS) and release of Co2+ from mLCP using three abiotic fluorophores. We also used Newport Green DCF dye to show that cobalt was taken up by the bacteria after mLCP exposure. Using transmission electron microscopy, we noted that the mLCP was not associated with the bacterial cell surface. In order for us to further probe the mechanism of interaction of mLCP, the bacteria were exposed to an equivalent dose of cobalt ions that dissolved from mLCP, which recapitulated the changes in viability when the bacteria were exposed to mLCP, and it also recapitulated the observed bacterial uptake of cobalt. Taken together, this implicates the release of cobalt ions and their subsequent uptake by the bacteria as the major toxicity mechanism of mLCP. The properties of the ENM govern the release rate of cobalt, but the toxicity does not arise from nanospecific effects-and importantly, the chemical composition of the ENM may dictate the oxidation state of the metal centers and thus limit ROS production.

Combination of rosemary extract and buffered vinegar inhibits Pseudomonas and Shewanella growth in common carp (Cyprinus carpio).

BACKGROUND: Aquaculture is the fastest growing food-production sector, and common carp (Cyprinus carpio) is one of the most cultivated fish species in the world. Due to its intrinsic characteristics, fish meat is highly susceptible to microbiological spoilage. Pseudomonas and Shewanella are the primary and secondary occurring microbiota during storage of fish meat, with significant contribution to spoilage with the formation of hydrolytic enzymes (lipases and proteases). RESULTS: With in vitro testing, we show that rosemary extract (Inolens4), buffered vinegar and their combination (SyneROX) exhibit antimicrobial effects against P. fragi, P. psychrophila, S. putrefaciens and S. xiaemensis at concentrations of 3.13 and 1.56 mg mL-1 . The combination was the most effective in inhibiting growth of selected bacteria in food model, and production of lipases and proteases during 9 days at 5 °C. In situ testing of antimicrobial dip treatment of carp meat determined that aerobic mesophilic, total psychrotrophic, Pseudomonas and hydrogen sulfide producer counts were reduced in all treatments, with the most prominent influence being shown by the combination and buffered vinegar. CONCLUSIONS: Our study highlights the importance of a multilevel assessment of the antimicrobial potential of biopreservatives under conditions comparable to those of the selected food. Investigation with bacteria and food model provided coherent and consistent data for the evaluation of the antimicrobial potential for carp meat. Combination of buffered vinegar (as active antimicrobial) and rosemary extract, with well-known and researched antioxidant properties but low in situ antimicrobial activity, represents a good potential for combined effect in preservation of fish meat. © 2020 Society of Chemical Industry.

Coupled Turbidity and Spectroscopy Problems: A Simple Algorithm for Volumetric Analysis of Optically Thin or Dilute, In Vitro Bacterial Cultures in Various Media.

An approach binary spectronephelometry (BSN) to perform real-time simultaneous noninvasive in situ physical and chemical analysis of bacterial cultures in fluid media is described. We choose to characterize cultures of Escherichia coli (NC), Pseudomonas aeruginosa (PA), and Shewanella oneidensis (SO) in the specific case of complex media whose Raman spectrum cannot be unambiguously assigned. Nevertheless, organism number density and a measure of the chemical makeup of the fluid medium can be monitored noninvasively, simultaneously, and continuously, despite changing turbidity and medium chemistry. The method involves irradiating a culture in fluid medium in an appropriate vessel (in this case a standard 1 cm cuvette) using a near infrared laser and collecting all the backscattered light from the cuvette, i.e., the Rayleigh-Mie line and the inelastically emitted light which includes unresolved Raman scattered light and fluorescence. Complex "legacy" media contain materials of biological origin whose chemical composition cannot be fully delineated. We independently calibrate this approach to a commonly used reference, optical density at 600 nm (OD600) for characterizing the number density of organisms. We suggest that the total inelastically emitted light could be a measure of the chemical state of a biologically based medium, e.g., lysogeny broth (LB). This approach may be useful in a broad range of basic and applied studies and enterprises that utilize bacterial cultures in any medium or container that permits optical probing in the single scattering limit.

Effects of phytic acid and lysozyme on microbial composition and quality of grass carp (Ctenopharyngodon idellus) fillets stored at 4 °C.

This study investigated the effect of phytic acid and lysozyme on the microbial composition and quality of grass carp (Ctenopharyngodon idellus) fillets stored at 4 °C. The control, 0.5 mg/mL lysozyme-treated fillets (T1), 0.5 mg/mL phytic acid-treated fillets (T2) and 0.25 mg/mL lysozyme + 0.25 mg/mL phytic acid-treated fillets (T3) were evaluated based on sensory assessment, biogenic amines, ATP-related compounds, total volatile basic nitrogen (TVB-N), and total viable counts (TVC). Changes in microbial composition were analyzed using high-throughput sequencing. Results showed that phytic acid and lysozyme treatment delayed the decrease in sensory scores, reduced the rate of degradation of IMP to Hx, inhibited the growth of microorganisms, and attenuated the increase in TVB-N and putrescine. Phytic acid exhibited better preservation effects than lysozyme and their combination was more effective than using either alone. High-throughput sequencing showed that Acinetobacter and Kocuria were the predominant bacteria in fresh grass carp, but Pseudomonas rose rapidly with storage time; Pseudomonas, Shewanella, and Aeromonas constituted the main spoilage bacteria of grass carp fillets. Lysozyme treatment significantly reduced the proportion of Shewanella and Acinetobacter, and phytic acid and the combination of phytic acid and lysozyme significantly reduced the proportion of Pseudomonas in spoiled grass carp fillets.

Microbial reduction of metal-organic frameworks enables synergistic chromium removal.

Redox interactions between electroactive bacteria and inorganic materials underpin many emerging technologies, but commonly used materials (e.g., metal oxides) suffer from limited tunability and can be challenging to characterize. In contrast, metal-organic frameworks exhibit well-defined structures, large surface areas, and extensive chemical tunability, but their utility as microbial substrates has not been examined. Here, we report that metal-organic frameworks can support the growth of the metal-respiring bacterium Shewanella oneidensis, specifically through the reduction of Fe(III). In a practical application, we show that cultures containing S. oneidensis and reduced metal-organic frameworks can remediate lethal concentrations of Cr(VI) over multiple cycles, and that pollutant removal exceeds the performance of either component in isolation or bio-reduced iron oxides. Our results demonstrate that frameworks can serve as growth substrates and suggest that they may offer an alternative to metal oxides in applications seeking to combine the advantages of bacterial metabolism and synthetic materials.

Transcriptional regulator ArcA mediates expression of oligopeptide transport systems both directly and indirectly in Shewanella oneidensis.

In γ-proteobacterial species, such as Escherichia coli, the Arc (anoxic redox control) two-component system plays a major role in mediating the metabolic transition from aerobiosis to anaerobiosis, and thus is crucial for anaerobic growth but dispensable for aerobic growth. In Shewanella oneidensis, a bacterium renowned for respiratory versatility, Arc (SoArc) primarily affects aerobic growth. To date, how this occurs has remained largely unknown although the growth defect resulting from the loss of DNA-binding response regulator SoArcA is tryptone-dependent. In this study, we demonstrated that the growth defect is in part linked to utilization of oligopeptides and di-tripeptides, and peptide uptake but not peptide degradation is significantly affected by the SoArcA loss. A systematic characterization of major small peptide uptake systems manifests that ABC peptide transporter Sap and four proton-dependent oligopeptide transporters (POTs) are responsible for transport of oligopeptides and di-tripeptides respectively. Among them, Sap and DtpA (one of POTs) are responsive to the SoarcA mutation but only dtpA is under the direct control of SoArcA. We further showed that both Sap and DtpA, when overproduced, improve growth of the SoarcA mutant. While the data firmly establish a link between transport of oligopeptides and di-tripeptides and the SoarcA mutation, other yet-unidentified factors are implicated in the growth defect resulting from the SoArcA loss.

Cold adaptation in the environmental bacterium Shewanella oneidensis is controlled by a J-domain co-chaperone protein network.

DnaK (Hsp70) is a major ATP-dependent chaperone that functions with two co-chaperones, a J-domain protein (JDP) and a nucleotide exchange factor to maintain proteostasis in most organisms. Here, we show that the environmental bacterium Shewanella oneidensis possesses a previously uncharacterized short JDP, AtcJ, dedicated to cold adaptation and composed of a functional J-domain and a C-terminal extension of 21 amino acids. We showed that atcJ is the first gene of an operon encoding also AtcA, AtcB and AtcC, three proteins of unknown functions. Interestingly, we found that the absence of AtcJ, AtcB or AtcC leads to a dramatically reduced growth at low temperature. In addition, we demonstrated that AtcJ interacts via its C-terminal extension with AtcC, and that AtcC binds to AtcB. Therefore, we identified a previously uncharacterized protein network that involves the DnaK system with a dedicated JDP to allow bacteria to survive to cold environment.