FEMS Microbiology Letters

Bradyrhizobia are widespread across the Australian continent, where they are essential to Australian ecosystems by helping legumes to compensate nutrient deficiencies and low fertility of Australian soils. Among the Bradyrhizobium genospecies identified during a survey of Australian native rhizobia communities in 1994-1995, genospecies L appeared to be only distantly related to any Bradyrhizobium lineages known at the time. We take advantage of the recent sequencing of the genome of strain BDV5419, the original strain corresponding to Bradyrhizobium genospecies L, to re-assess this lineage taxonomic status. We characterized further strain BDV5419 based on morpho-physiological traits and determined its phylogenetic relationships with the type strains of the 88 currently known Bradyrhizobium species based on sequence comparisons of SSU rRNA genes and complete genomes. The digital DNA-DNA hybridization relatedness with any type strain was less than 33% and both SSU rRNA gene and genome phylogenies confirmed that this strain does not belong to any formerly described species within the Bradyrhizobium genus. Whereas its position within the lineage encompassing the B. elkanii and B. jicamae supergroups is unresolved in the SSU rDNA phylogeny, strain BDV5419 appears to be one of most basal lineages of the B. elkanii supergroup in the genome comparison. All data thus support the description of the novel species Bradyrhizobium hardenbergiae sp. nov. which type strain is BDV5419T (= CFBP 9111T = LMG 32897T), isolated from a nodule of Hardenbergia violaceae in Black Mountain Nature Reserve, in Canberra, ACT, Australia.

The major food-borne pathogen Campylobacter jejuni employs chemotactic motility to colonise the avian gut, and also as a virulence mechanism in human diarrhoeal disease. In Escherichia coli CheY activity is modulated by CheZ, a phosphatase originally thought to be absent in C. jejuni. The Hp0170 protein of Helicobacter pylori is a distant homologue of CheZ and, as C. jejuni Cj0700 is homologous to HP0170, Cj0700 could also act as a CheZ orthologue in Campylobacter. Both the C. jejuni CheV and CheA proteins also contain a response regulator (RR) domain that may be phosphorylated. Cj0700 would therefore be predicted to dephosphorylate C. jejuni CheY and possibly also the CheV and CheA RR domains. A mutant ({Delta}cj0700) and complement ({Delta}cj0700, cj0046::cj0700) were constructed in C.jejuni strains NCTC11168, NCTC11828 and 81-176. On semisolid agar the {Delta}cj0700 mutant strain showed reduced motility relative to wild-type and this phenotype was reversed in the complemented strain. In pull down and bacterial two hybrid assays, expressed Cj0700 was able to interact with CheY, CheA-RR and CheV. Cj0700 is able to dephosphorylate the RR domain of CheY and CheA-RR, but less efficiently, CheV. These findings verify that Cj0700 plays a role in C. jejuni chemotaxis through phosphatase activity with respect to CheY, and is hence likely to be a CheZ orthologue. Cj0700 also partially modulates the phosphorylation level of the RR domain on CheA and CheV, although the functional consequences of this interaction require further investigation.

NifA is a {sigma}54 activator that turns on bacterial nitrogen fixation under reducing conditions and when fixed cellular nitrogen levels are low. The redox sensing mechanism in -proteobacterial NifA is poorly understood. In this work, we examine if a Cys pair that is part of a C(X)5C motif and located immediately upstream of NifAs DNA binding domain is involved in redox sensing in NifA from the -proteobacterium Gluconacetobacter diazotrophicus (Gd). We hypothesize that the Cys residues redox state may directly influence the DNA binding domains DNA binding affinity and/or alter the proteins oligomeric sate. Two DNA binding domain constructs were generated, a longer construct (2C-DBD), consisting of the DNA binding domain with the upstream Cys pair, and a shorter construct (NC-DBD) that lacks the Cys pair. The Kd of NC-DBD for its cognate DNA sequence (nifH-UAS) is equal to 20.0 M. The Kd of 2C-DBD for nifH-UAS when the Cys pair is oxidized is 34.5 M. Reduction of the disulfide bond does not change the DNA binding affinity. Additional experiments indicate that the redox state of the Cys residues does not influence the secondary structure or oligomerization state of the NifA DNA binding domain. Together, these results demonstrate that the Cys pair upstream of the DNA binding domain of Gd-NifA does not regulate DNA binding or domain dimerization in a redox dependent manner. This suggests that other Cys residues in NifA, such as those located in the central AAA+ domain, are responsible for redox sensing.

Currently, there are three recognized rhizobial genera belonging to the beta branch of the proteobacteria; Trinickia, Paraburkholderia, and Cupriavidus. These beta-rhizobia have been found associated with legume species mainly within the Mimosoideae and Papillonoideae. Most diversity, evolutionary, and functional studies have focused on Paraburkholderia, whereas few have addressed the diversity and evolution of symbiosis in the Cupriavidus genus. The present work aimed to provide an actual view of the symbiotic Cupriavidus diversity and to analyse the origin and evolution of their symbiotic genes. Using whole-genome information for phylogenetic reconstruction, we showed that the described symbiotic Cupriavidus strains belong to five distinct lineages, although they are intermixed with non-symbiotic species. The high synteny and sequence conservation of symbiotic genes suggest a common origin of acquisition for all rhizobial Cupriavidus described so far. However, we observed very low sequence conservation among (mega)plasmids carrying the symbiotic island, excluding the existence of a conserved symbiotic plasmid within beta-rhizobia. We can conclude that up to now there are five rhizobial species within the Cupriavidus genus, and we predict the description of new symbiotic species in the near future.

For Escherichia coli, an adaptive response to temperatures just above the minimum temperature of growth, 8{degrees}C, includes a change in morphology from rods to small rods. A study was initiated to determine the requirement of nucleoid-associated protein FIS for growth and genome compaction in the small rods at low temperature. Growth and nucleoid staining analyses revealed that the fis null mutant displayed decreased growth and initially formed filaments containing decondensed nucleoids at 12{degrees}C, indicating that FIS facilitates production of small rods with condensed nucleoids at low temperature. However, characterized by biphasic growth at low temperature, the fis null mutant exhibited increased growth, cell division, and nucleoid condensation following a lag phase. Furthermore, compacted circular-shaped nucleoids were formed near the onset of the second growth phase. Therefore E. coli responds to the absence of FIS by inducing an adaptive mechanism that causes a shift towards nucleoid condensation resulting in genome compaction in small rods. Furthermore, the deletion of sulA (encodes DNA damaged-induced cell division inhibitor SulA) in the fis null mutant resulted in suppression of the filamentous morphology. This indicates that the absence of FIS with nucleoid decondensation led to DNA damage, inducing cell division inhibition by SulA.

Recently Hordt et al. 2020 proposed to merge Ochrobactrum and Brucella genera based on up to date phylogenomic evidence and overall genomic divergence among Brucella-Ochrobactrum clade. This led to the description of the new combinations Brucella ciceri comb. nov., basonym: Ochrobactrum ciceri Imran et al. 2010 and Brucella intermedia comb. nov., basonym: Ochrobactrum intermedium Velasco et al. 1998. However, the type species for Brucella ciceri DSM 22292T and Brucella intermedia LMG 3301T show whole-genome coherence at the species level (ANI = 98.21 %, Mash D = 0.0154006, dDDH relatedness >70%), suggesting that may belong to the same genomospecies. Also, both taxa formed a single clade in the phylogenomic tree based on single-copy gene sequences. Previously reported phenotypic data offer a context where both taxa are highly related supporting this synonymy. Therefore, Brucella ciceri should be reclassified as later heterotypic synonyms of Brucella intermedia, which has priority. The species description is consequently amended.

Membrane cardiolipin (CL) phospholipids play a fundamental role in the adaptation of bacteria to various environmental conditions, including saline stress. Here, we constructed deletion mutants of two CL synthetase genes, clsA and clsB, in the rhizobacterium Pseudomonas fluorescens UM270, and evaluated their role in plant growth promotion under salt stress. P. fluorescens UM270 {Delta}clsA and {Delta}clsB mutants showed a significant reduction in CL synthesis compared to the UM270 wild-type strain (58% {Delta}clsA and 53% {Delta}clsB), and their growth rate was not affected, except when grown at 100 and 200 mM NaCl. Additionally, the root colonization capacity of both mutant strains was impaired compared with that of the wild type. Concomitant with the deletion of clsA and clsB, some physiological changes were observed in the UM270 {Delta}clsA and {Delta}clsB mutants, such as a reduction in indole acetic acid and biofilm production. By contrast, an increase in siderophore biosynthesis was observed. Further, inoculation of the UM270 wild-type strain in tomato plants (Lycopersicon esculentum Saladette) grown under salt stress conditions (100 and 200 mM NaCl) resulted in an increase in root and shoot length, chlorophyll content, and dry weight. On the contrary, when each of the mutants ({Delta}clsA and {Delta}clsB) were inoculated in tomato plants, a reduction in root length was observed when grown at 200 mM NaCl, but the shoot length, chlorophyll content, and total plant dry weight parameters were significantly reduced under normal or saline conditions (100 and 200 mM NaCl), compared to UM270 wild-type-inoculated plants. In conclusion, these results suggest that CL synthesis in P. fluorescens UM270 plays an important role in the promotion of tomato plant growth under normal conditions, but to a greater extent, under salt-stress conditions.

The present study described the comparative genomic analysis of the validly named species of the genus Pseudomonas to define the taxonomic assignment. Genomic information for 208 type strains was available in the NCBI genome database at the time of conducting this analysis. The ANI, AAI and in silico DNA DNA hybridization (isDDH) data were higher than the threshold values for the twelve strains with their closely related type species. Whole genome comparisons shared 97 - 99 % average nucleotide identity, 97.85 to 99.19 % average amino acid identity and 72.80 to 90.40 % digital DNA DNA hybridization values. Further, the phylogenomic analysis based on the core genome confirmed that P. humi CCA1 and P. citronellolis LMG 18378, P. zeshuii KACC 15471 and P. luteola NBRC 103146, P. oryzihabitans DSM 6835 and P. psychrotolerans DSM 15758, P. nitroreducens DSM 14399 and P. nitritireducens WZBFD3-5A2, P. fluvialis CCM 8778 and P. pharmacofabricae ZYSR67-Z, P. panacis DSM 18529 and P. marginalis DSM 13124 formed a monophyletic clade. Thus, we proposed six type species viz., P. humi CCA1, P. zeshuii KACC 15471, P. psychrotolerans DSM 15758, P. nitritireducens WZBFD3 5A2, P. pharmacofabricae ZYSR67 Z and P. panacis DSM 18529 are the later heterotypic synonym of P. citronellolis Lang 2007, P. luteola, P. oryzihabitans, P. nitroreducens Lang 2007, P. fluvialis and P. marginalis (Brown 1918) Stevens 1925 (Approved Lists 1980), respectively considering the priority date of publication.

Bacteria accumulate small, organic compounds, called compatible solutes, via uptake from the environment or biosynthesis from available precursors to maintain the turgor pressure of the cell in response to osmotic stress. Vibrio parahaemolyticus has biosynthesis pathways for the compatible solutes ectoine (ectABCasp_ect) and glycine betaine (betIBAproXWV), four betaine-carnitine-choline transporters (bcct1-bcct4) and a second ProU transporter (proVWX). Most of these systems are induced in high salt. CosR, a MarR-type regulator, which is divergently transcribed from bcct3, was previously shown to be a direct repressor of ectABCasp_ect in Vibrio species. In this study, we investigated the role of CosR in glycine betaine biosynthesis and compatible solute transporter gene regulation. Expression analyses demonstrated that betIBAproXWV, bcct1, bcct3, and proVWX are repressed in low salinity. Examination of an in-frame cosR deletion mutant shows induced expression of these systems in the mutant at low salinity compared to wild-type. DNA binding assays demonstrate that purified CosR binds directly to the regulatory region of each system. In Escherichia coli GFP reporter assays, we demonstrate that CosR directly represses transcription of betIBAproXWV, bcct3, and proVWX. Similar to V. harveyi, we show betIBAproXWV is positively regulated by the LuxR homolog OpaR. Bioinformatics analysis demonstrates that CosR is widespread within the genus, present in over 50 species. In several species, the cosR homolog was clustered with the betIBAproXWV operon, which again suggests the importance of this regulator in glycine betaine biosynthesis. Incidentally, in four Aliivibrio species that contain ectoine biosynthesis genes, we identified another MarR-type regulator, ectR, clustered with these genes, which suggests the presence of a novel ectoine regulator. Homologs of EctR in this genomic context were present in A. fischeri, A. finisterrensis, A. sifiae and A. wodanis. ImportanceVibrio parahaemolyticus can accumulate compatible solutes via biosynthesis and transport, which allow the cell to survive in high salinity conditions. There is little need for compatible solutes under low salinity conditions, and biosynthesis and transporter systems are repressed. However, the mechanism of this repression is not fully elucidated. CosR plays a major role in the repression of multiple compatible solute systems in V. parahaemolyticus as a direct negative regulator of ectoine and glycine betaine biosynthesis systems and four transporters. Homology analysis suggests that CosR functions in this manner in many other Vibrio species. In Aliivibrio species, we identified a new MarR family regulator EctR that clusters with the ectoine biosynthesis genes.

Saccharomyces cerevisiae can alter its morphology to a filamentous form associated with unipolar budding in response to environmental stressors. Induction of filamentous growth is suggested under nitrogen deficiency in response to alcoholic signalling molecules through a quorum sensing mechanism. To investigate this claim, we analysed the budding pattern of S. cerevisiae cells over time under low nitrogen while concurrently measuring cell density and extracellular metabolite concentration. We found that the proportion of cells displaying unipolar budding increased between local cell densities of 4.8x106 and 5.3x107 cells/ml. However, the observed increase in unipolar budding could not be reproduced when cells were prepared at the critical cell density and in conditioned media. Removing the nutrient restriction by growth under high nitrogen conditions also resulted in an increase in unipolar budding between local cell densities of 5.2x106 and 8.2x107 cells/ml, but there were differences in metabolite concentration compared to the low nitrogen conditions. This suggests that neither cell density, metabolite concentration, nor nitrogen deficiency were necessary or sufficient to increase the proportion of unipolar budding cells. It is therefore unlikely that quorum sensing is the mechanism controlling the switch to filamentous growth in S. cerevisiae. Only a high concentration of the putative signalling molecule, 2-phenylethanol resulted in an increase in unipolar budding, but this concentration was not physiologically relevant. We suggest that the compound 2-phenylethanol acts through a toxicity mechanism, rather than quorum sensing, to induce filamentous growth. IMPORTANCEInvestigating dimorphism in the model organism Saccharomyces cerevisiae has been instrumental in understanding the signalling pathways that control hyphal growth and virulence in human pathogenic fungi. Quorum sensing was proposed to signal morphogenesis in S. cerevisiae populations. This mechanism requires the switch to filamentous growth to occur at a critical quorum sensing molecule concentration corresponding to a critical cell density. However, evidence for this mechanism is sparse and limited by the use of non-physiologically relevant concentrations of signalling metabolites. Our study designed a methodology to address this gap and may be applied to further studies of dimorphism in other types of yeasts. A significant implication of our findings is that morphogenesis does not occur in S. cerevisiae via a quorum sensing mechanism, and this important definition needs to be corrected. Mechanistic studies to understand dimorphism in yeasts, by considering metabolite concentrations, will further shed light onto this important cellular behaviour.

Genetic diversity of populations is essential for generating phenotypic variation to allow a flexible response to a shift in environmental conditions. Therefore, in populations of genetically identical individuals grown in the lab, you would predict that phenotypic heterogeneity would be small. However, we isolated two subpopulations of genetically identical individuals from an exponentially growing batch culture of the microalga Chlamydomonas reinhardtii using Percoll step-gradients. The culture fractionated into a low-density, Top fraction and a high-density, Bottom fraction. These subpopulations displayed several phenotypic differences, including size, protein content, the amount of chlorophyll per cell, and photosynthetic performance. Because of the variation in pigment content and photosynthetic performance, we tested the hypothesis that there are differences in their tolerance to light stress. Following high-light stress, the Bottom subpopulation was more resistant to photodamage, had a greater capacity for light dissipation, and had a minimal photoacclimation response to high light, compared to the Top subpopulation. The Bottom population also had a greater resistance to exogenously induced singlet oxygen stress mediated by rose bengal. We hypothesize that these subpopulations are derived from stochastic mechanism where the Bottom subpopulation has activated a general high-light stress response pathway as part of a "bet-hedging" strategy that could give it a fitness advantage with a shift towards a light-stress environment.

Bacillus cereus G9241 was isolated from a welder who survived a pulmonary anthrax-like disease. Strain G9241 carries two virulence plasmids, pBCX01 and pBC210, as well as an extrachromosomal prophage, pBFH_1. pBCX01 has 99.6% sequence identity to pXO1 carried by Bacillus anthracis and encodes the tripartite anthrax toxin genes and atxA, a mammalian virulence transcriptional regulator. This work looks at how the presence of pBCX01 and temperature may affect the lifestyle of B. cereus G9241 using a transcriptomic analysis and by studying spore formation, an important part of the B. anthracis lifecycle. Here we report that pBCX01 has a stronger effect on gene transcription at the mammalian infection relevant temperature of 37{degrees}C in comparison to 25{degrees}C. At 37{degrees}C, the presence of pBCX01 appears to have a negative effect on genes involved in cell metabolism, including biosynthesis of amino acids, whilst positively affecting the transcription of many transmembrane proteins. The study of spore formation showed B. cereus G9241 sporulated rapidly in comparison to the B. cereus sensu stricto type strain ATCC 14579, particularly at 37{degrees}C. The carriage of pBCX01 did not affect this phenotype suggesting that other genetic elements were driving rapid sporulation. An unexpected finding of this study was that pBFH_1 is highly expressed at 37{degrees}C in comparison to 25{degrees}C and pBFH_1 expression leads to the production of Siphoviridae-like phage particles in the supernatant of B. cereus G9241. This study provides an insight on how the extrachromosomal genetic elements in B. cereus G9241 has an influence in bacterial phenotypes.

Sugar-induced cell death (SICD) remains an intriguing but poorly studied phenomenon in the physiology of Saccharomyces cerevisiae. Recently, it was shown that SICD development largely depends on the redirection of glucose fluxes between glycolysis and the pentose phosphate pathway (PPP). In particular, inhibition of glycolysis by iodoacetamide (IAA) was observed to reduce SICD levels. This study is devoted to further investigation of the relationship between SICD and the functionality of the two PPP branches. It was shown that deletion of the ZWF1 gene does not affect the decrease in SICD levels in IAA-treated cells. This allows us to conclude that the oxidative branch of the PPP is not involved in the suppression of SICD/ROS. Deletion of the GLR1 gene and attenuation of the TRR1 gene also did not restore SICD levels in cells after IAA treatment. The obtained results indicate that the level of reduced glutathione or thioredoxin does not affect SICD genesis. The addition of 5 mM ribose-5-phosphate (R5P) to the incubation medium led to suppression of SICD by 79%. At the same time, the addition of 5 mM ribose + 5 mM Pi suppressed SICD by only 20%. Suppression of SICD by 5 mM R5P in the{Delta} pho3 strain (83%) excludes the mechanism of extracellular dephosphorylation of R5P to ribose, its subsequent transport into the cell, and re-phosphorylation inside the cell. Furthermore, more than 70% suppression of SICD in the{Delta} end3 strain with 5 mM R5P excludes endocytosis as a mechanism of R5P import into the cell. The observed effect of R5P can be explained by the moonlighting function of some unknown protein. Thus, SICD development in S. cerevisiae cells depends on the final product of the non-oxidative PPP--R5P.

A gene encoding OvoA, a key enzyme involved in the biosynthesis of ovothiol, was excised form the genome of Leishmania mexicana promastigotes using CRISPR/cas mediated gene editing. The role of the enzyme in synthesising ovothiol was confirmed since both ovothiol A and ovothiol B were lost from the metabolome of the modified cells. The OvoA knockout line had similar growth kinetics to wild-type progenitor cells and, moreover, most of the changes in metabolism that accompanied the transition of log stage growth to stationary phase were mirrored in the KO line. Significant differences, however, were observed in the ratio of the reduced and oxidised forms of the other major low molecular weight thiols, glutathione and trypanothione, indicative of a role of these other thiols in maintaining reduced ovothiol and demonstrating an interconnected network of low molecular weight thiols in these cells. The OvoA knockout cells remained infective to macrophages where promastigotes transformed to amastigote forms in a manner similar to wild-type. The knockout line was tested for sensitivity to a range of current anti-leishmanial drugs and oxidative and nitrosative stresses. While generally the absence of ovothiol caused little or no change in sensitivity to these stress-inducing agents, enhanced sensitivity to amphotericin B was noted. Author summaryOvothiol is a low molecular weight histidine-derived thiol first described in sea urchin eggs, and later found in many organisms, including the protozoa of the order Kinetoplastida, that includes human pathogens such as the Leishmania species that cause leishmaniasis. Thiol metabolism in the Kinetoplastidae has been studied in some detail, particularly with regard to an unusual bis-glutathione, spermidine conjugate named trypanothione that takes on many of the roles performed by glutathione in most other organisms. Roles for ovothiol in Leishmania have not been previously defined, although potential roles in defence against oxidative stress have been hypothesised. A gene encoding the first enzyme of the pathway involved in ovothiol production, OvoA, was excised from the Leishmania mexicana genome. Its role in ovothiol synthesis was confirmed as ovothiol was absent from the mutants. Little changed, however, with respect to the phenotype of these cells, including their proliferation rate, their ability to infect macrophages or their sensitivity to a range of stress inducing agents. These included several leishmanicidal drugs, oxidative and nitrosative stresses. For amphotericin B, however, the Ovothiol lacking cells were more sensitive than wild-type indicating some role in defence against the impact of this drug.

Strain LLG6346-3.1T, isolated from the thallus of the brown alga Ericaria zosteroides collected in Mediterranean Sea near Bastia in Corsica, France, was characterized using a polyphasic method. Cells were Gram-stain-negative, strictly aerobic, non-flagellated, motile by gliding, rod-shaped and grew optimally at 30-33 {degrees}C, at pH 8-8.5 and with 4-5 % NaCl. Strain LLG6346-3.1T used the seaweed polysaccharide alginic acid as sole carbon source which was vigorously liquefied. Phylogenetic analyses showed that the bacterium is affiliated to the genus Zobellia (family Flavobacteriaceae, class Flavobacteriia). Strain LLG6346-3.1T exhibited 16S rRNA gene sequence similarity values of 98.5 and 98.3 % to the type strains of Zobellia russellii and Zobellia roscoffensis respectively, and of 97.4-98.2 % to other species of the genus Zobellia. The DNA G+C content of strain LLG6346-3.1T was determined to be 38.28 mol%. Digital DNA-DNA hybridization predictions by the ANI and GGDC methods between strain LLG6346-3.1T and other members of the genus Zobellia showed values of 76-88 %, and below 37 %, respectively. The phenotypic, phylogenetic and genomic analyses show that strain LLG6346-3.1T is distinct from species of the genus Zobellia with validly published names and that it represents a novel species of the genus Zobellia, for which the name Zobellia alginoliquefaciens sp. nov. is proposed. The type strain is LLG6346-3.1T (RCC 7657T = LLG 32918T).

Stenotrophomonas goyi sp. nov. has been isolated from a contaminated algal culture (Chlamydomonas reinhardtii). Its genome has been fully sequenced (4,487,389 base pairs) and a tentative annotation is provided (4,147 genes). The genome information suggests that S. goyi sp. nov. is unable to use sulfate and nitrate as sulfur and nitrogen sources, respectively. Growth tests have confirmed the dependence of the sulfur-containing amino acids methionine and cysteine. The potential biotechnological interest of this bacteria is discussed here and in a related research paper (Fakhimi et al., 2023b).

Besides an industrial pollutant, 2,4-dinitrophenol (DNP) has been used illegally as a weight loss drug that had claimed human lives. Little is known about the metabolism of DNP, particularly among Gram-negative bacteria. In this study, two non-contiguous genetic loci of Paraburkholderia (formerly Burkholderia) sp. strain KU-46 genome were identified and four key initial genes (dnpA, dnpB, and dnpC1C2) were characterized to provide molecular and biochemical evidence for the degradation of DNP via the formation of 4-nitrophenol (NP), a pathway that is unique among DNP utilizing bacteria. Reverse transcription PCR analysis indicated that the dnpA gene encoding the initial hydride transferase (28 kDa), and the dnpB gene encoding a nitrite-eliminating enzyme (33 kDa), are inducible by DNP and the two genes are organized in an operon. Purified DnpA and DnpB from overexpression clones in Escherichia coli effected the transformation of DNP to NP via the formation of hydride-Meisenheimer complex of DNP. The function of DnpB appears new since all homologs of DnpB sequences in the protein database are annotated as putative nitrate ABC transporter substrate-binding proteins. The gene cluster responsible for the degradation of DNP after NP formation was designated dnpC1C2DXFER. DnpC1 and DnpC2 were functionally characterized as the respective FAD reductase and oxygenase components of the two-component NP monooxygenase. Both NP and 4-nitrocatechol were shown to be substrates, producing hydroquinone and hydroxyquinol, respectively. Elucidation of the hqdA1A2BCD gene cluster allows the delineation of the final degradation pathway of hydroquinone to {beta}-ketoadipate prior to its entry to the tricarboxylic acid cycle.\n\nImportanceThis study fills a gap in our knowledge and understanding of the genetic basis and biochemical pathway for the degradation of 2,4-dinitrophenol (DNP) in Gram-negative bacteria, represented by the prototypical Paraburkholderia sp. strain KU-46 that metabolizes DNP through the formation of 4-nitrophenol, a pathway unseen by other DNP utilizers. The newly cloned genes could serve as DNA probes in biomonitoring as well as finding application in new biocatalyst development to access green chemicals. By and large, knowledge of the diverse strategies used by microorganisms to degrade DNP will contribute to the development of bioremediation solutions since DNP is an industrial pollutant used widely in the chemical industry for the synthesis of pesticides, insecticides, sulfur dyes, wood preservatives, and explosives, etc. (119 words)

Glycosaminoglycans (GAGs), comprising uronic acids and amino sugars, are widely distributed in human tissues such as the intestine and oral cavity. Various bacteria colonize these tissues by assimilating GAGs. 4-Deoxy-L-threo-5-hexosulose uronate (DHU) is produced during the degradation of GAGs. Pectin, abundant in plants, is also degraded into DHU. DHU is metabolized in a stepwise manner by the isomerase KduI or DhuI, followed by the reductase KduD or DhuD. Previous studies have shown that the two genes encoding isomerase and reductase (kduI-kduD and dhuD-dhuI, respectively) are designated in an operon. Therefore, it was believed that dhuD-dhuI and kduI-kduD operons evolved independently. Nevertheless, the discovery of a hybrid kduI-dhuD operon raised questions regarding the evolution of these operons. This study was conducted to investigate the factors driving the diversity of operons through a pan-genomic phylogenetic analysis across 3550 bacterial strains. Seven types of DHU metabolism-related operons were identified. The phylum Bacteroidota possesses a hybrid-type kduI-dhuD operon rather than kduI-kduD or kduD-kduI operon. The phylum Bacillota, but not Pseudomonadota or Bacteroidota, possesses the dhuD-dhuI operon; however, dhuI-dhuD operon was not detected in any bacterial strain. Although DHU is generated from the degradation of oligomerized GAG by an unsaturated glucuronyl hydrolase (UGL), the UGL gene was found in strains positive for kduD-kduI, dhuD-dhuI, kduI-dhuD, and dhuD-kduI operons at high ratios, indicating that the acquisition of these operons is advantageous for colonization on human hosts. (230 words) IMPORTANCEGlycosaminoglycans (GAGs), crucial components of the extracellular matrix, play a vital role in host infection by pathogenic bacteria as well as in the colonization of the host by commensal bacteria. The isomerases KduI and DhuI are nonhomologous isofunctional enzymes. The dhuD-dhuI operon is well conserved within certain phyla and appears to have a strong association with GAG metabolism. In contrast, the kduI system is more widely distributed across various species. Based on the possession ratios of genes encoding each enzyme that produces 4-deoxy-L-threo-5-hexosulose uronate, this study indicated that the substrates targeted by each metabolic system vary depending on the specific operon type.

All bacterial epibiotic predators are rich in secondary metabolites and most genera rich in secondary metabolites have demonstrable predatory abilities. Therefore it is likely that an antibiotic resistant and thereby predation resistant species may benefit not only by escaping predation but also by utilizing nutrients released by lysis of prey cells by predatory bacteria. The resistant organisms may enjoy greater fitness benefits than the predator since they get the benefit without investing in the predation machinery. In our experiment, a marine isolate of Streptomyces atrovirens showed good predatory activity on a range of species including Staphylococcus aureus and Proteus vulgaris. Escherichia coli was resistant to predation by this species. On slide culture with water agar when the predator, S. aureus and E. coli were grown together S. aureus population declined whereas the predation resistant E. coli increased their population as compared to controls. However the growth of E. coli did not affect growth of the predator unfavorably. This strengthens the possibility that evolution of antibiotic resistance not only gave a selective advantage of escaping predation, it also would have increased the fitness of the resistant organism by promoting growth on nutrients released from the prey cells lysed by the predator. When the predator was grown with S. aureus and P. vulgaris as prey, S. aureus declined rapidly whereas P. vulgaris was spared. This suggests that the predator appears to show preference towards prey and in that case even a partial or relative resistance may give substantial advantage to a population.

Purine moieties are essential for many functions within the eukaryotic cell, including energy, signaling and nucleic acid synthesis. While purine starvation is known to induce stress resistance in eukaryotic model organism budding yeast Saccharomyces cerevisiae, it remains unclear whether the physiological response is related to disruption of synthesis pathway in particular position or it is uniform across all genetic deficiencies within the de novo adenine biosynthesis pathway. It is also not known how purine starved cells perceive purine shortage - weather they share the same signaling elements with nitrogen starvation or not. MethodsWe characterised physiology of strains with deletions in adenine biosynthesis pathway when cultivated in full or purine deficient and compared to cell physiological parameters when cultivated in nitrogen deficient media. We tested stress tolerance, carbon flux, cell cycle arrest and did transcription profiling (RNA-seq). ResultsOur findings demonstrate that purine starvation-induced stress resistance is significantly modulated by the specific step at which the pathway is interrupted. Transcriptional analysis revealed that purine starvation in many aspects phenocopies nitrogen starvation, particularly - in both starvations strong downregulation of ribosome related genes occurs. In the same time several metabolic features which differ from N- and ade- starvations: pentose phosphate pathway is specifically upregulated within ade4{Delta}-ade2{Delta} and downregulated in N-cells. Notably, the expression of stress-responsive genes such as HSP12, HSP26, and GRE1 varied between mutants, suggesting that the accumulation of pathway intermediates (e.g., AIR in ade2{Delta}) or the absence of downstream precursors (AICAR) alters the perception of starvation especially in the case of ade16{Delta}ade17{Delta} strain. ConclusionsMetabolic and stress-tolerance phenotypes of purine auxotrophs are not merely a result of purine depletion but seems that the response is signalled via the same pathways, like TOR1. The results suggest that strains having mutations within various positions of the purine pathway "perceive" purine limitation a bit differently - especially when we compare the end of the pathway with the other mutants. Different phenotypic outcomes of the occasional purine depletion might give preferences for organisms which have mutations in the beginning rather at the end of the pathway. Besides, our findings might have implications in the design of synthetic pathways and the use of auxotrophic markers in yeast research.