Inactivation of Pseudomonas putida KT2440 pyruvate dehydrogenase relieves catabolite repression and improves the usefulness of this strain for degrading aromatic compounds.

Pyruvate dehydrogenase (PDH) catalyses the irreversible decarboxylation of pyruvate to acetyl-CoA, which feeds the tricarboxylic acid cycle. We investigated how the loss of PDH affects metabolism in Pseudomonas putida. PDH inactivation resulted in a strain unable to utilize compounds whose assimilation converges at pyruvate, including sugars and several amino acids, whereas compounds that generate acetyl-CoA supported growth. PDH inactivation also resulted in the loss of carbon catabolite repression (CCR), which inhibits the assimilation of non-preferred compounds in the presence of other preferred compounds. Pseudomonas putida can degrade many aromatic compounds, most of which produce acetyl-CoA, making it useful for biotransformation and bioremediation. However, the genes involved in these metabolic pathways are often inhibited by CCR when glucose or amino acids are also present. Our results demonstrate that the PDH-null strain can efficiently degrade aromatic compounds even in the presence of other preferred substrates, which the wild-type strain does inefficiently, or not at all. As the loss of PDH limits the assimilation of many sugars and amino acids and relieves the CCR, the PDH-null strain could be useful in biotransformation or bioremediation processes that require growth with mixtures of preferred substrates and aromatic compounds.

Two new species and a new record of stygobitic freshwater snails (Gastropoda, Cochliopidae) from Candela, Coahuila, northern Mexico.

We describe two new species and a new record of stygobitic gastropods from small groundwater-fed springs in Candela, Coahuila, northern Mexico. Phreatomascogos garciasaucedoi n. sp. is described based on shell morphology and is the second species of this formerly monotypic genus. According to the current classification, we have transferred this genus to Cochliopidea. Phreatodrobia candelensis n. sp. is described, and represents the first record of the genus in Mexico extending its known range more than one hundred kilometers to the south. Also found with the new stygosnails was Coahuilix hubbsi Taylor, 1966, which was previously known only as an endemic species from the neighboring Cuatro Cinegas valley. The reported new subterranean snails are restricted in their distributions to two small water sources only a few meters long which flow directly into a touristic zone with swimming pools and other recreation areas. Using NatureServe Ranking, both new species were assigned as critically imperiled. The very limited distribution and negative anthropogenic impacts within the sites should draw special conservation attention for the reported stygobionts.

What are the signals that control catabolite repression in Pseudomonas?

Metabolically versatile bacteria exhibit a global regulatory response known as carbon catabolite repression (CCR), which prioritizes some carbon sources over others when all are present in sufficient amounts. This optimizes growth by distributing metabolite fluxes, but can restrict yields in biotechnological applications. The molecular mechanisms and preferred substrates for CCR vary between bacterial groups. Escherichia coli prioritizes glucose whereas Pseudomonas sp. prefer certain organic acids or amino acids. A significant issue in understanding (and potentially bypassing) CCR is the lack of information about the signals that trigger this regulatory response. In E. coli, several key compounds act as flux sensors, governing the flow of metabolites through catabolic pathways and preventing imbalances. These flux sensors can also modulate the CCR response. It has been suggested that the order of substrate preference is determined by carbon uptake flux rather than substrate identity. For Pseudomonas, much less information is available, as the signals that induce CCR are poorly understood. This article briefly discusses the available evidence on the signals that trigger CCR and the questions that remain to be answered in Pseudomonas.

Extraordinary long-stem confers resistance of intrinsic terminators to processive antitermination.

Many prokaryotic operons encode a processive antitermination (P-AT) system. Transcription complexes associated with an antitermination factor can bypass multiple transcription termination signals regardless of their sequences. However, to avoid compromising transcriptional regulation of downstream regions, the terminator at the end of the operon needs to be resistant to antitermination. So far, no studies on the mechanism of resistance to antitermination have been reported. The recently discovered conAn P-AT system is composed of two components that are encoded at the start of many conjugation operons on plasmids of Gram-positive bacteria. Here we report the identification of a conAn-resistant terminator, named TerR, in the conjugation operon of the Bacillus subtilis plasmid pLS20, re-defining the end of the conjugation operon. We investigated the various characteristics of TerR and show that its extraordinary long stem is the determining feature for resistance to antitermination. This is the first P-AT resistance mechanism to be reported.

Bioprospecting microbes and enzymes for the production of pterocarpans and coumestans.

The isoflavonoid derivatives, pterocarpans and coumestans, are explored for multiple clinical applications as osteo-regenerative, neuroprotective and anti-cancer agents. The use of plant-based systems to produce isoflavonoid derivatives is limited due to cost, scalability, and sustainability constraints. Microbial cell factories overcome these limitations in which model organisms such as Saccharomyces cerevisiae offer an efficient platform to produce isoflavonoids. Bioprospecting microbes and enzymes can provide an array of tools to enhance the production of these molecules. Other microbes that naturally produce isoflavonoids present a novel alternative as production chassis and as a source of novel enzymes. Enzyme bioprospecting allows the complete identification of the pterocarpans and coumestans biosynthetic pathway, and the selection of the best enzymes based on activity and docking parameters. These enzymes consolidate an improved biosynthetic pathway for microbial-based production systems. In this review, we report the state-of-the-art for the production of key pterocarpans and coumestans, describing the enzymes already identified and the current gaps. We report available databases and tools for microbial bioprospecting to select the best production chassis. We propose the use of a holistic and multidisciplinary bioprospecting approach as the first step to identify the biosynthetic gaps, select the best microbial chassis, and increase productivity. We propose the use of microalgal species as microbial cell factories to produce pterocarpans and coumestans. The application of bioprospecting tools provides an exciting field to produce plant compounds such as isoflavonoid derivatives, efficiently and sustainably.

The acetoin assimilation pathway of Pseudomonas putida KT2440 is regulated by overlapping global regulatory elements that respond to nutritional cues.

Many microorganisms produce and excrete acetoin (3-hydroxy-2-butanone) when growing in environments that contain glucose or other fermentable carbon sources. This excreted compound can then be assimilated by other bacterial species such as pseudomonads. This work shows that acetoin is not a preferred carbon source of Pseudomonas putida, and that the induction of genes required for its assimilation is down-modulated by different, independent, global regulatory systems when succinate, glucose or components of the LB medium are also present. The expression of the acetoin degradation genes was found to rely on the RpoN alternative sigma factor and to be modulated by the Crc/Hfq, Cyo and PTSNtr regulatory elements, with the impact of the latter three varying according to the carbon source present in addition to acetoin. Pyruvate, a poor carbon source for P. putida, did not repress acetoin assimilation. Indeed, the presence of acetoin significantly improved growth on pyruvate, revealing these compounds to have a synergistic effect. This would provide a clear competitive advantage to P. putida when growing in environments in which all the preferred carbon sources have been depleted and pyruvate and acetoin remain as leftovers from the fermentation of sugars by other microorganisms.

Expression of the ISPpu9 transposase of Pseudomonas putida KT2440 is regulated by two small RNAs and the secondary structure of the mRNA 5'-untranslated region.

Insertion sequences (ISs) are mobile genetic elements that only carry the information required for their own transposition. Pseudomonas putida KT2440, a model bacterium, has seven copies of an IS called ISPpu9 inserted into repetitive extragenic palindromic sequences. This work shows that the gene for ISPpu9 transposase, tnp, is regulated by two small RNAs (sRNAs) named Asr9 and Ssr9, which are encoded upstream and downstream of tnp, respectively. The tnp mRNA has a long 5'-untranslated region (5'-UTR) that can fold into a secondary structure that likely includes the ribosome-binding site (RBS). Mutations weakening this structure increased tnp mRNA translation. Asr9, an antisense sRNA complementary to the 5'-UTR, was shown to be very stable. Eliminating Asr9 considerably reduced tnp mRNA translation, suggesting that it helps to unfold this secondary structure, exposing the RBS. Ectopic overproduction of Asr9 increased the transposition frequency of a new ISPpu9 entering the cell by conjugation, suggesting improved tnp expression. Ssr9 has significant complementarity to Asr9 and annealed to it in vitro forming an RNA duplex; this would sequester it and possibly facilitate its degradation. Thus, the antisense Asr9 sRNA likely facilitates tnp expression, improving transposition, while Ssr9 might counteract Asr9, keeping tnp expression low.

Multiple Layered Control of the Conjugation Process of the Bacillus subtilis Plasmid pLS20.

Bacterial conjugation is the main horizontal gene transfer route responsible for the spread of antibiotic resistance, virulence and toxin genes. During conjugation, DNA is transferred from a donor to a recipient cell via a sophisticated channel connecting the two cells. Conjugation not only affects many different aspects of the plasmid and the host, ranging from the properties of the membrane and the cell surface of the donor, to other developmental processes such as competence, it probably also poses a burden on the donor cell due to the expression of the large number of genes involved in the conjugation process. Therefore, expression of the conjugation genes must be strictly controlled. Over the past decade, the regulation of the conjugation genes present on the conjugative Bacillus subtilis plasmid pLS20 has been studied using a variety of methods including genetic, biochemical, biophysical and structural approaches. This review focuses on the interplay between RcopLS20, RappLS20 and Phr*pLS20, the proteins that control the activity of the main conjugation promoter P c located upstream of the conjugation operon. Proper expression of the conjugation genes requires the following two fundamental elements. First, conjugation is repressed by default and an intercellular quorum-signaling system is used to sense conditions favorable for conjugation. Second, different layers of regulation act together to repress the P c promoter in a strict manner but allowing rapid activation. During conjugation, ssDNA is exported from the cell by a membrane-embedded DNA translocation machine. Another membrane-embedded DNA translocation machine imports ssDNA in competent cells. Evidences are reviewed indicating that conjugation and competence are probably mutually exclusive processes. Some of the questions that remain unanswered are discussed.

An Improved Protocol for Agrobacterium-Mediated Transformation in Subterranean Clover (Trifolium subterraneum L.).

Subterranean clover (Trifolium subterraneum) is the most widely grown annual pasture legume in southern Australia. With the advent of advanced sequencing and genome editing technologies, a simple and efficient gene transfer protocol mediated by Agrobacterium tumefaciens was developed to overcome the hurdle of genetic manipulation in subterranean clover. In vitro tissue culture and Agrobacterium transformation play a central role in testing the link between specific genes and agronomic traits. In this paper, we investigate a variety of factors affecting the transformation in subterranean clover to increase the transformation efficiency. In vitro culture was optimised by including cefotaxime during seed sterilisation and testing the best antibiotic concentration to select recombinant explants. The concentrations for the combination of antibiotics obtained were as follows: 40 mg L-1 hygromycin, 100 mg L-1 kanamycin and 200 mg L-1 cefotaxime. Additionally, 200 mg L-1 cefotaxime increased shoot regeneration by two-fold. Different plant hormone combinations were tested to analyse the best rooting media. Roots were obtained in a medium supplemented with 1.2 µM IAA. Plasmid pH35 containing a hygromycin-resistant gene and GUS gene was inoculated into the explants with Agrobacterium tumefaciens strain AGL0 for transformation. Overall, the transformation efficiency was improved from the 1% previously reported to 5.2%, tested at explant level with Cefotaxime showing a positive effect on shooting regeneration. Other variables in addition to antibiotic and hormone combinations such as bacterial OD, time of infection and incubation temperature may be further tested to enhance the transformation even more. This improved transformation study presents an opportunity to increase the feeding value, persistence, and nutritive value of the key Australian pasture.

A new global regulator that facilitates the co-metabolization of polyaromatic hydrocarbons and other nutrients in Novosphingobium.

In an article in this issue of Environmental Microbiology, Segura et al. report the identification of an unusual global regulator in Novosphingobium sp. HR1a, a metabolically versatile bacterial strain isolated from the rhizosphere able to assimilate a wide range of polyaromatic hydrocarbons (PAHs). Physiological and transcriptomic assays suggest that this regulator, named PahT, activates the expression of genes involved in the assimilation of PAHs, and of compounds such as sugars and acetate, facilitating their co-metabolism. This effect is the opposite to the carbon catabolite repression strategy that allows metabolically versatile bacteria to favour the use of some compounds over others. PahT was found to stimulate sugar uptake and metabolization in the presence and absence of PAHs and to facilitate microaerobic respiration if PAHs were present. A survey of the genomes of several Sphingomonadaceae members showed that PahT is not present in all strains of this family, but that it is strongly associated with PAH degradation genes. Since not all PAH-degrading strains contain pahT, it seems that PahT is not essential for PAH degradation but likely provides a selective advantage to PAH-degrading strains in environments such as the rhizosphere where other potential carbon sources are available.

Influence of the Crc global regulator on substrate uptake rates and the distribution of metabolic fluxes in Pseudomonas putida KT2440 growing in a complete medium.

When the soil bacterium Pseudomonas putida grows in a complete medium, it prioritizes the assimilation of preferred carbon sources, optimizing its metabolism and growth. This regulatory process is orchestrated by the Crc and Hfq proteins. The present work examines the changes that occur in metabolic fluxes when the crc gene is inactivated and cells grow exponentially in LB complete medium. Analyses were performed at three different moments during exponential growth, examining the assimilation rates for the compounds present in LB, changes in the proteome, and the changes in metabolic fluxes predicted by the iJN1411 metabolic model for P. putida KT2440. During the early exponential phase, consumption rates for sugars, many organic acids and most amino acids were higher in a Crc-null strain than in the wild type, leading to an overflow of the metabolic pathways and the leakage of pyruvate and acetate. These accelerated consumption rates decreased during the mid-exponential phase, when cells mostly used sugars and alanine. At later times, pyruvate was recovered from the medium and utilized. The higher consumption rates of the Crc-null strain reduced the growth rate. The lack of the Crc/Hfq regulatory system thus led to unbalanced metabolism with poorly optimized metabolic fluxes.

Pseudomonas putida KT2440 metabolism undergoes sequential modifications during exponential growth in a complete medium as compounds are gradually consumed.

Pseudomonas putida is a soil bacterium with a versatile and robust metabolism. When confronted with mixtures of carbon sources, it prioritizes the utilization of the preferred compounds, optimizing metabolism and growth. This response is particularly strong when growing in a complex medium such as LB. This work examines the changes occurring in P. putida KT2440 metabolic fluxes, while it grows exponentially in LB medium and sequentially consumes the compounds available. Integrating the uptake rates for each compound at three different moments during the exponential growth with the changes observed in the proteome, and with the metabolic fluxes predicted by the iJN1411 metabolic model for this strain, allowed the metabolic rearrangements that occurred to be determined. The results indicate that the bacterium changes significantly the configuration of its metabolism during the early, mid and late exponential phases of growth. Sugars served as an energy source during the early phase and later as energy and carbon source. The configuration of the tricarboxylic acids cycle varied during growth, providing no energy in the early phase, and turning to a reductive mode in the mid phase and to an oxidative mode later on. This work highlights the dynamism and flexibility of P. putida metabolism.

Novel regulatory mechanism of establishment genes of conjugative plasmids.

The principal route for dissemination of antibiotic resistance genes is conjugation by which a conjugative DNA element is transferred from a donor to a recipient cell. Conjugative elements contain genes that are important for their establishment in the new host, for instance by counteracting the host defense mechanisms acting against incoming foreign DNA. Little is known about these establishment genes and how they are regulated. Here, we deciphered the regulation mechanism of possible establishment genes of plasmid p576 from the Gram-positive bacterium Bacillus pumilus. Unlike the ssDNA promoters described for some conjugative plasmids, the four promoters of these p576 genes are repressed by a repressor protein, which we named Reg576. Reg576 also regulates its own expression. After transfer of the DNA, these genes are de-repressed for a period of time until sufficient Reg576 is synthesized to repress the promoters again. Complementary in vivo and in vitro analyses showed that different operator configurations in the promoter regions of these genes lead to different responses to Reg576. Each operator is bound with extreme cooperativity by two Reg576-dimers. The X-ray structure revealed that Reg576 has a Ribbon-Helix-Helix core and provided important insights into the high cooperativity of DNA recognition.

CRISPR-Cas systems: ushering in the new genome editing era.

In recent years there has been great progress with the implementation and utilization of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) systems in the world of genetic engineering. Many forms of CRISPR-Cas9 have been developed as genome editing tools and techniques and, most recently, several non-genome editing CRISPR-Cas systems have emerged. Most of the CRISPR-Cas systems have been classified as either Class I or Class II and are further divided among several subtypes within each class. Research teams and companies are currently in dispute over patents for these CRISPR-Cas systems as numerous powerful applications are concurrently under development. This mini review summarizes the appearance of CRISPR-Cas systems with a focus on the predominant CRISPR-Cas9 system as well as the classifications and subtypes for CRISPR-Cas. Non-genome editing uses of CRISPR-Cas are also highlighted and a brief overview of the commercialization of CRISPR is provided.

Influence of the Hfq and Crc global regulators on the control of iron homeostasis in Pseudomonas putida.

Metabolically versatile bacteria use catabolite repression control to select their preferred carbon sources, thus optimizing carbon metabolism. In pseudomonads, this occurs through the combined action of the proteins Hfq and Crc, which form stable tripartite complexes at target mRNAs, inhibiting their translation. The activity of Hfq/Crc is antagonised by small RNAs of the CrcZ family, the amounts of which vary according to carbon availability. The present work examines the role of Pseudomonas putida Hfq protein under conditions of low-level catabolite repression, in which Crc protein would have a minor role since it is sequestered by CrcZ/CrcY. The results suggest that, under these conditions, Hfq remains operative and plays an important role in iron homeostasis. In this scenario, Crc appears to participate indirectly by helping CrcZ/CrcY to control the amount of free Hfq in the cell. Iron homeostasis in pseudomonads relies on regulatory elements such as the Fur protein, the PrrF1-F2 sRNAs, and several extracytoplasmic sigma factors. Our results show that the absence of Hfq is paralleled by a reduction in PrrF1-F2 small RNAs. Hfq thus provides a regulatory link between iron and carbon metabolism, coordinating the iron supply to meet the needs of the enzymes operational under particular nutritional regimes.

Differential expression of the three Alcanivorax borkumensis SK2 genes coding for the P450 cytochromes involved in the assimilation of hydrocarbons.

Alcanivorax borkumensis, a marine bacterium highly specialized in degrading linear and branched alkanes, plays a key ecological role in the removal of marine oil spills. It contains several alternative enzyme systems for terminal hydroxylation of alkanes, including three P450 cytochromes (P450-1, P450-2 and P450-3). The present work shows cytochrome P450-1 to be expressed from the promoter of the upstream gene fdx. Promoter Pfdx was more active when C8 -C18 n-alkanes or pristane were assimilated than when pyruvate was available. The product of ABO_0199 (named CypR) was identified as a transcriptional activator of Pfdx . The inactivation of cypR impaired growth on tetradecane, showing the importance of the fdx-P450-1 and/or cypR genes. P450-2 expression was low-level and constitutive under all conditions tested, while that of P450-3 from promoter P450-3 was much higher when cells assimilated pristane than when n-alkanes or pyruvate were available. However, the inactivation of P450-3 had no visible impact on pristane assimilation. Cyo terminal oxidase, a component of the electron transport chain, was found to stimulate promoter PP450-3 activity, but it did not affect promoters Pfdx or PP450-2 . A. borkumensis, therefore, appears to carefully coordinate the expression of its multiple hydrocarbon degradation genes using both specific and global regulatory systems.

Glucose uptake in Azotobacter vinelandii occurs through a GluP transporter that is under the control of the CbrA/CbrB and Hfq-Crc systems.

Azotobacter vinelandii, a strict aerobic, nitrogen fixing bacterium in the Pseudomonadaceae family, exhibits a preferential use of acetate over glucose as a carbon source. In this study, we show that GluP (Avin04150), annotated as an H+-coupled glucose-galactose symporter, is the glucose transporter in A. vinelandii. This protein, which is widely distributed in bacteria and archaea, is uncommon in Pseudomonas species. We found that expression of gluP was under catabolite repression control thorugh the CbrA/CbrB and Crc/Hfq regulatory systems, which were functionally conserved between A. vinelandii and Pseudomonas species. While the histidine kinase CbrA was essential for glucose utilization, over-expression of the Crc protein arrested cell growth when glucose was the sole carbon source. Crc and Hfq proteins from either A. vinelandii or P. putida could form a stable complex with an RNA A-rich Hfq-binding motif present in the leader region of gluP mRNA. Moreover, in P. putida, the gluP A-rich Hfq-binding motif was functional and promoted translational inhibition of a lacZ reporter gene. The fact that gluP is not widely distributed in the Pseudomonas genus but is under control of the CbrA/CbrB and Crc/Hfq systems demonstrates the relevance of these systems in regulating metabolism in the Pseudomonadaceae family.

Effect of Crc and Hfq proteins on the transcription, processing, and stability of the Pseudomonas putida CrcZ sRNA.

In Pseudomonas putida, the Hfq and Crc proteins regulate the expression of many genes in response to nutritional and environmental cues, by binding to mRNAs that bear specific target motifs and inhibiting their translation. The effect of these two proteins is antagonized by the CrcZ and CrcY small RNAs (sRNAs), the levels of which vary greatly according to growth conditions. The crcZ and crcY genes are transcribed from promoters PcrcZ and PcrcY, respectively, a process that relies on the CbrB transcriptional activator and the RpoN σ factor. Here we show that crcZ can also be transcribed from the promoter of the immediate upstream gene, cbrB, a weak constitutive promoter. The cbrB-crcZ transcript was processed to render a sRNA very similar in size to the CrcZ produced from promoter PcrcZ The processed sRNA, termed CrcZ*, was able to antagonize Hfq/Crc because, when provided in trans, it relieved the deregulated Hfq/Crc-dependent hyperrepressing phenotype of a ΔcrcZΔcrcY strain. CrcZ* may help in attaining basal levels of CrcZ/CrcZ* that are sufficient to protect the cell from an excessive Hfq/Crc-dependent repression. Since a functional sRNA can be produced from PcrcZ, an inducible strong promoter, or by cleavage of the cbrB-crcZ mRNA, crcZ can be considered a 3'-untranslated region of the cbrB-crcZ mRNA. In the absence of Hfq, the processed form of CrcZ was not observed. In addition, we show that Crc and Hfq increase CrcZ stability, which supports the idea that these proteins can form a complex with CrcZ and protect it from degradation by RNases.