T6SS translocates a micropeptide to suppress STING-mediated innate immunity by sequestering manganese.

Cellular ionic concentrations are a central factor orchestrating host innate immunity, but no pathogenic mechanism that perturbs host innate immunity by directly targeting metal ions has yet been described. Here, we report a unique virulence strategy of Yersinia pseudotuberculosis (Yptb) involving modulation of the availability of Mn2+, an immunostimulatory metal ion in host cells. We showed that the Yptb type VI secretion system (T6SS) delivered a micropeptide, TssS, into host cells to enhance its virulence. The mutant strain lacking TssS (ΔtssS) showed substantially reduced virulence but induced a significantly stronger host innate immune response, indicating an antagonistic role of this effector in host antimicrobial immunity. Subsequent studies revealed that TssS is a Mn2+-chelating protein and that its Mn2+-chelating ability is essential for the disruption of host innate immunity. Moreover, we showed that Mn2+ enhances the host innate immune response to Yptb infection by activating the stimulator of interferon genes (STING)-mediated immune response. Furthermore, we demonstrated that TssS counteracted the cytoplasmic Mn2+ increase to inhibit the STING-mediated innate immune response by sequestering Mn2+ Finally, TssS-mediated STING inhibition sabotaged bacterial clearance in vivo. These results reveal a previously unrecognized bacterial immune evasion strategy involving modulation of the bioavailability of intracellular metal ions and provide a perspective on the role of the T6SS in pathogenesis.

A Yersinia T6SS Effector YezP Engages the Hemin Uptake Receptor HmuR and ZnuABC for Zn2+ Acquisition.

Metal ions are essential nutrients for all life forms, and restriction of metal ion availability is an effective host defense against bacterial infection. Meanwhile, bacterial pathogens have developed equally effective means to secure their metal ion supply. The enteric pathogen Yersinia pseudotuberculosis was found to uptake zinc using the T6SS4 effector YezP, which is essential for Zn2+ acquisition and bacterial survival under oxidative stress. However, the mechanism of this zinc uptake pathway has not been fully elucidated. Here, we identified the hemin uptake receptor HmuR for YezP, which can mediate import of Zn2+ into the periplasm by the YezP-Zn2+ complex and demonstrated that YezP functions extracellularly. This study also confirmed that the ZnuCB transporter is the inner membrane transporter for Zn2+ from the periplasm to cytoplasm. Overall, our results reveal the complete T6SS/YezP/HmuR/ZnuABC pathway, wherein multiple systems are coupled to support zinc uptake by Y. pseudotuberculosis under oxidative stress. IMPORTANCE Identifying the transporters involved in import of metal ions under normal physiological growth conditions in bacterial pathogens will clarify its pathogenic mechanism. Y. pseudotuberculosis YPIII, a common foodborne pathogen that infects animals and humans, uptake zinc via the T6SS4 effector YezP. However, the outer and inner transports involved in Zn2+ acquisition remain unknown. The important outcomes of this study are the identification of the hemin uptake receptor HmuR and inner membrane transporter ZnuCB that import Zn2+ into the cytoplasm via the YezP-Zn2+ complex, and elucidation of the complete Zn2+ acquisition pathway consisting of T6SS, HmuRSTUV, and ZnuABC, thereby providing a comprehensive view of T6SS-mediated ion transport and its functions.

Siderophore-Mediated Iron Acquisition Enhances Resistance to Oxidative and Aromatic Compound Stress in Cupriavidus necator JMP134.

Many bacteria secrete siderophores to enhance iron uptake under iron-restricted conditions. In this study, we found that Cupriavidus necator JMP134, a well-known aromatic pollutant-degrading bacterium, produces an unknown carboxylate-type siderophore named cupriabactin to overcome iron limitation. Using genome mining, targeted mutagenesis, and biochemical analysis, we discovered an operon containing six open reading frames (cubA-F) in the C. necator JMP134 genome that encodes proteins required for the biosynthesis and uptake of cupriabactin. As the dominant siderophore of C. necator JMP134, cupriabactin promotes the growth of C. necator JMP134 under iron-limited conditions via enhanced ferric iron uptake. Furthermore, we demonstrated that the iron concentration-dependent expression of the cub operon is mediated by the ferric uptake regulator (Fur). Physiological analyses revealed that the cupriabactin-mediated iron acquisition system influences swimming motility, biofilm formation, and resistance to oxidative and aromatic compound stress in C. necator JMP134. In conclusion, we identified a carboxylate-type siderophore named cupriabactin, which plays important roles in iron scavenging, bacterial motility, biofilm formation, and stress resistance.IMPORTANCE Since siderophores have been widely exploited for agricultural, environmental, and medical applications, the identification and characterization of new siderophores from different habitats and organisms will have great beneficial applications. Here, we identified a novel siderophore-producing gene cluster in C. necator JMP134. This gene cluster produces a previously unknown carboxylate siderophore, cupriabactin. Physiological analyses revealed that the cupriabactin-mediated iron acquisition system influences swimming motility, biofilm formation, and oxidative stress resistance. Most notably, this system also plays important roles in increasing the resistance of C. necator JMP134 to stress caused by aromatic compounds, which provide a promising strategy to engineer more efficient approaches to degrade aromatic pollutants.

Rhizobium gei sp. nov., a bacterial endophyte of Geum aleppicum.

A bacterial strain, designated as ZFJT-2T, was isolated from the stem of Geum aleppicum Jacq. collected from Taibai Mountain in Shaanxi Province, north-west China. Cells of strain ZFJT-2T were Gram-stain-negative, strictly aerobic, rod-shaped and motile by means of a single polar flagellum. The major fatty acids were summed feature 8 (comprising C18 : 1ω7c and/or C18 : 1ω6c), C16 : 0, 11-methyl C18 : 1ω7c and summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω6c), and the DNA G+C content was 58.3 mol% (HPLC). Phylogenetic analyses based on 16S rRNA gene sequences showed that strain ZFJT-2T was a member of the genus Rhizobium and was most closely related to Rhizobium giardinii KACC 10720T (98.6 % similarity) and Rhizobium herbae CCBAU 83011T (98.5 %). The low levels of sequence similarity found between the atpD, recA and glnII gene sequences of strain ZFJT-2T and those of recognized species of the genus Rhizobium (no more than 94.4, 87.2 and 89.5 %, respectively) indicated that it may represent a separate species of the genus Rhizobium. The DNA-DNA relatedness values for strain ZFJT-2T with respect to R. giardinii KACC 10720T and R. herbae CCBAU 83011T were 17.6 and 41.9 %, respectively. On the basis of phenotypic, phylogenetic and genotypic data, strain ZFJT-2T is considered to represent a novel species of the genus Rhizobium, for which the name Rhizobium gei sp. nov. is proposed. The type strain is ZFJT-2T (=CCTCC AB 2013015T=KCTC 32301T=LMG 27603T).

Sphingomonas hylomeconis sp. nov., isolated from the stem of Hylomecon japonica.

A yellow-pigmented bacterium, designated strain GZJT-2T, was isolated from the stem of Hylomecon japonica (Thunb.) Prantl et Kündig collected from Taibai Mountain in Shaanxi Province, north-west China. Cells of strain GZJT-2T were Gram-reaction-negative, strictly aerobic, rod-shaped, non-spore-forming and non-motile. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain GZJT-2T was a member of the genus Sphingomonas, with sequence similarities of 92.1-96.9 % to type strains of recognized species of the genus Sphingomonas (92.1 % to Sphingomonas oligoaromativorans SY-6T and 96.9 % to Sphingomonas oligophenolica JCM 12082T). Strain GZJT-2T contained ubiquinone-10 (Q-10) as the predominant respiratory quinone and sym-homospermidine as the major polyamine. The major cellular fatty acids were summed feature 8 (comprising C18 : 1ω7c and/or C18 : 1ω6c), summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω6c), C16 : 0 and C14 : 0 2-OH. Phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylcholine, sphingoglycolipid, four unidentified phospholipids, an unidentified aminolipid and four unidentified lipids were detected in the polar lipid profile. The DNA G+C content was 62.5 ± 0.3 mol%. On the basis of data from phenotypic, phylogenetic and DNA-DNA relatedness studies, strain GZJT-2T is considered to represent a novel species of the genus Sphingomonas, for which the name Sphingomonas hylomeconis sp. nov. is proposed. The type strain is GZJT-2T ( = CCTCC AB 2013304T = KCTC 42739T).

Rhizobacter bergeniae sp. nov., isolated from the root of Bergenia scopulosa.

A yellowish-pigmented bacterium, designated strain PLGR-1(T), was isolated from the root of Bergenia scopulosa collected from Taibai Mountain in Shaanxi Province, north-west China, and was subjected to a taxonomic study by using a polyphasic approach. Cells of strain PLGR-1(T) were Gram-stain-negative, strictly aerobic, rod-shaped, non-spore-forming and motile with a single polar flagellum. Growth occurred at 7-33 °C (optimum, 25-28 °C), at pH 5.0-10.0 (optimum, pH 6.0-7.0) and with 0-0.5 % (w/v) NaCl (optimum, 0 %). The predominant respiratory quinone was ubiquinone-8 (Q-8) and the major cellular fatty acids were summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω6c), C16 : 0 and summed feature 8 (comprising C18 : 1ω7c and/or C18 : 1ω6c). The major polyamines were putrescine and 2-hydroxyputrescine and the major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The DNA G+C content was 69.8 mol%. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain PLGR-1(T) belonged to the class Betaproteobacteria and formed a tight phyletic lineage with members of the genus Rhizobacter. Strain PLGR-1(T) was most closely related to Rhizobacter dauci DSM 11587(T) and Rhizobacter fulvus DSM 19916(T), with 16S rRNA gene sequence similarities of 98.5 and 98.0 %, respectively. The DNA-DNA relatedness values between strain PLGR-1(T) and the type strains of Rhizobacter dauci and Rhizobacter fulvus were 46.3 and 14.7 %, respectively. Based on the phenotypic, phylogenetic and genotypic data, strain PLGR-1(T) is considered to represent a novel species of the genus Rhizobacter, for which the name Rhizobacter bergeniae sp. nov. is proposed. The type strain is PLGR-1(T) ( = CCTCC AB 2013018(T) = KCTC 32299(T) = LMG 27607(T)).

Sphingomonas gei sp. nov., isolated from roots of Geum aleppicum.

A yellow-pigmented bacterium, designated strain ZFGT-11(T), was isolated from roots of Geum aleppicum Jacq. collected from Taibai Mountain in Shaanxi Province, north-west China, and was subjected to a taxonomic study by using a polyphasic approach. Cells of strain ZFGT-11(T) were Gram-stain-negative, strictly aerobic rods that were surrounded by a thick capsule and were motile by means of a single polar flagellum. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain ZFGT-11(T) was a member of the genus Sphingomonas and was closely related to Sphingomonas naasensis KACC 16534(T) (97.6% similarity), Sphingomonas kyeonggiense JCM 18825(T) (96.8%), Sphingomonas asaccharolytica IFO 15499(T) (96.7%) and Sphingomonas leidyi DSM 4733(T) (96.6%). The predominant respiratory quinone was ubiquinone-10 (Q-10) and the major cellular fatty acids were summed feature 8 (comprising C(18 : 1)ω7c and/or C(18 : 1)ω6c), C(17 : 1)ω6c, C(14 : 0) 2-OH, C(16 : 0) and C(15 : 0) 2-OH. The major polyamine of strain ZFGT-11(T) was sym-homospermidine. Phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylcholine, sphingoglycolipid, two unidentified aminoglycolipids, two unidentified phospholipids and two unidentified lipids were detected in the polar lipid profile. The DNA G+C content was 66.8 mol%. DNA-DNA relatedness for strain ZFGT-11(T) with respect to its closest phylogenetic relative S. naasensis KACC 16534(T) was 26.2±4.8% (mean±SD). On the basis of data from the present polyphasic taxonomic study, strain ZFGT-11(T) is considered to represent a novel species of the genus Sphingomonas , for which the name Sphingomonas gei sp. nov. is proposed. The type strain is ZFGT-11(T) ( = CCTCC AB 2013306(T) = KCTC 32449(T) = LMG 27608(T)).

Sphingobium endophyticus sp. nov., isolated from the root of Hylomecon japonica.

A yellow-pigmented bacterium, designated strain GZGR-4(T), was isolated from the root of Hylomecon japonica (Thunb.) Prantl et Kündig collected from Taibai Mountain in Shaanxi Province, north-west China. Cells of strain GZGR-4(T) were Gram-negative, rod-shaped, strictly aerobic, non-endospore-forming and non-motile. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain GZGR-4(T) is a member of the genus Sphingobium, exhibiting the highest sequence similarity to Sphingobium aromaticiconvertens DSM 12677(T) (97.3 %). 16S rRNA gene sequence similarities between strain GZGR-4(T) and the type strains of other Sphingobium species with validly published names ranged from 93.4-96.5 %. The predominant respiratory quinone of strain GZGR-4(T) was ubiquinone-10 (Q-10) and the major cellular fatty acids were summed feature 8 (comprising C18:1 ω7c and/or C18:1 ω6c), summed feature 3 (comprising C16:1 ω7c and/or C16:1 ω6c), C16:0 and C14:0 2-OH. Spermidine was the major polyamine. The polar lipid profile consisted of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, sphingoglycolipid, one unidentified phosphoglycolipid, one unidentified phospholipid, one unidentified aminolipid and one unidentified lipid. The DNA G+C content was 63.6 mol%. DNA-DNA relatedness for strain GZGR-4(T) with respect to its closest phylogenetic relative S. aromaticiconvertens DSM 12677(T) was 22.6 ± 5.3 %. On the basis of the polyphasic taxonomic data presented, strain GZGR-4(T) is considered to represent a novel species of the genus Sphingobium, for which the name Sphingobium endophyticus sp. nov. is proposed. The type strain is GZGR-4(T) (=CCTCC AB 2013305(T) = KCTC 32447(T)).

Asticcacaulis endophyticus sp. nov., a prosthecate bacterium isolated from the root of Geum aleppicum.

A strictly aerobic, light-yellow-coloured, stalked bacterium, designated strain ZFGT-14(T), was isolated from the root of Geum aleppicum Jacq. collected from Taibai Mountain in Shaanxi province, north-west China, and was subjected to a taxonomic study using a polyphasic approach. This novel isolate grew at 7-33 °C (optimum 25-28 °C) and pH 6.0-10.0 (optimum pH 7.0-8.0). Flexirubin-type pigments were not produced. Cells were Gram-stain-negative, rod-shaped and motile with a single polar flagellum. The predominant respiratory quinone was Q-10. The major cellular fatty acids were summed feature 8 (comprising C18 : 1ω7c/C18 : 1ω6c), C16 : 0, C19 : 0 cyclo ω8c and summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω6c) and the major polar lipids were phosphatidylglycerol and glycolipids. The DNA G+C content was 57.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain ZFGT-14(T) was most closely related to the genus Asticcacaulis and had low sequence similarity (95.0-95.9 %) with all species with validly published names within the genus Asticcacaulis. Based on the phenotypic, phylogenetic and genotypic data, strain ZFGT-14(T) is considered to represent a novel species of the genus Asticcacaulis, for which the name Asticcacaulis endophyticus sp. nov. is proposed. The type strain is ZFGT-14(T) ( = CCTCC AB 2013012(T) = KCTC 32296(T) = LMG 27605(T)).

Pseudoxanthomonas gei sp. nov., a novel endophytic bacterium isolated from the stem of Geum aleppicum.

A Gram stain-negative, strictly aerobic, rod-shaped, non-motile and deep-yellow-coloured bacterial strain, designated ZFJR-3(T), was isolated from the stem of Geum aleppicum Jacq. collected from Taibai Mountain in Shaanxi Province, north-west China, and characterized by using a polyphasic approach. The novel isolate grew optimally at 25-28 °C and in the absence of NaCl. Flexirubin-type pigments were produced. The predominant respiratory quinone was ubiquinone-8 (Q-8) and the major cellular fatty acids were iso-C15:0 (29.2 %), iso-C16:0 (18.5 %), summed feature 9 (comprising iso-C17:1 ω9c and/or C16:0 10-methyl; 8.8 %), C16:1 ω7c alcohol (8.8 %), iso-C11:0 3-OH (6.9 %) and iso-C11:0 (6.8 %). The DNA G+C content was 66.1 mol %. The only polyamine was spermidine and the major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain ZFJR-3(T) belongs to the genus Pseudoxanthomonas and was most closely related to Pseudoxanthomonas yeongjuensis KCTC 22757(T) (16S rRNA gene sequence similarity, 99.0 %) and Pseudoxanthomonas sacheonensis KCTC 22080(T) (98.0 %). The levels of 16S rRNA gene sequence similarity with respect to other Pseudoxanthomonas species with validly published names were less than 96.5 %. DNA-DNA relatedness values for strain ZFJR-3(T) with respect to its closely related neighbours P. yeongjuensis KCTC 22757(T) and P. sacheonensis KCTC 22080(T) were 48.7 and 36.3 %, respectively. Based on the phenotypic, phylogenetic and genotypic data, strain ZFJR-3(T) is considered to represent a novel species of the genus Pseudoxanthomonas, for which the name Pseudoxanthomonas gei sp. nov. is proposed. The type strain is ZFJR-3(T) (=CCTCC AB 2013020(T) =KCTC 32298(T)).

Solirubrobacter taibaiensis sp. nov., isolated from a stem of Phytolacca acinosa Roxb.

A white-coloured bacterium, designated strain GTJR-20(T), was isolated from a stem of Phytolacca acinosa Roxb. collected from Taibai Mountain in Shaanxi Province, north-west China, and was subjected to a taxonomic study by using a polyphasic approach. The novel isolate was found to grow optimally at 28-30 °C, at pH 7.5-8.0 and in the absence of NaCl. Cells were observed to be Gram-stain positive, strictly aerobic, rod-shaped and non-motile. The predominant respiratory quinone was identified as MK-7(H4) and the major cellular fatty acids were identified as iso-C16:0 (35.8 %), C18:1 ω9c (17.7 %), C17:1 ω6c (11.0 %), C17:1 ω8c (7.8 %) and C18:3 ω6c (6, 9, 12) (7.2 %). The DNA G+C content was determined to be 71.6 mol %. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain GTJR-20(T) is a member of the genus Solirubrobacter and is closely related to Solirubrobacter phytolaccae GTGR-8(T) (16S rRNA gene sequence similarity, 98.4 %), Solirubrobacter soli KCTC 12628(T) (97.8 %), Solirubrobacter pauli KCTC 9974(T) (97.7 %) and Solirubrobacter ginsenosidimutans KCTC 19420(T) (97.6 %). No other recognized bacterial species showed more than 94.6 % 16S rRNA gene sequence similarity to the novel isolate. DNA-DNA relatedness values for strain GTJR-20(T) with respect to its closely related neighbours S. phytolaccae GTGR-8(T), S. soli KCTC 12628(T), S. pauli KCTC 9974(T) and S. ginsenosidimutans KCTC 19420(T) were 48.3 ± 8.6, 21.3 ± 5.2, 36.8 ± 6.2 and 36.0 ± 5.5 %, respectively. Based on the phenotypic, phylogenetic and genotypic data, strain GTJR-20(T) is considered to represent a novel species of the genus Solirubrobacter, for which the name Solirubrobacter taibaiensis sp. nov. is proposed. The type strain is GTJR-20(T) (=CCTCC AB 2013308(T) = KCTC 29222(T)).

Rhizobium smilacinae sp. nov., an endophytic bacterium isolated from the leaf of Smilacina japonica.

During a study of endophytic bacteria from traditional Chinese medicinal plants, a bacterial strain, designated PTYR-5(T), was isolated from the leaf of Smilacina japonica A. Gray collected from Taibai Mountain in Shaanxi Province, north-west China. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain PTYR-5(T) is a member of the genus Rhizobium, exhibiting the highest sequence similarities to R. cellulosilyticum LMG 23642(T) (97.2%), R. huautlense LMG 18254(T) (97.2%) and R. alkalisoli CCBAU 01393(T) (97.1%). The levels of 16S rRNA gene sequence similarity with respect to other Rhizobium species with validly published names were less than 97.0%. Phylogenies of the housekeeping genes atpD, recA and glnII confirmed its distinct position, showing low similarity with respect to those of recognized Rhizobium species (no more than 94.1, 90.0 and 88.0% similarity, respectively). The DNA-DNA relatedness values of strain PTYR-5(T) with R. cellulosilyticum LMG 23642(T), R. huautlense LMG 18254(T) and R. alkalisoli CCBAU 01393(T) were 33.6, 21.4 and 29.5 %, respectively. Based on phenotypic, phylogenetic and genotypic data, strain PTYR-5(T) is considered to represent a novel species of the genus Rhizobium, for which the name Rhizobium smilacinae sp. nov. is proposed. The type strain is PTYR-5(T) (=CCTCC AB 2013016(T)=KCTC 32300(T)=LMG 27604(T)).

OxyR-regulated T6SS functions in coordination with siderophore to resist oxidative stress.

The formation of reactive oxygen species is harmful and can destroy intracellular macromolecules such as lipids, proteins, and DNA, even leading to bacterial death. To cope with this situation, microbes have evolved a variety of sophisticated mechanisms, including antioxidant enzymes, siderophores, and the type VI secretion system (T6SS). However, the mechanism of oxidative stress resistance in Cupriavidus pinatubonensis is unclear. In this study, we identified Reut_A2805 as an OxyR ortholog in C. pinatubonensis, which positively regulated the expression of T6SS1 by directly binding to its operon promoter region. The study revealed that OxyR-regulated T6SS1 combats oxidative stress by importing iron into bacterial cells. Moreover, the T6SS1-mediated outer membrane vesicles-dependent iron acquisition pathway played a crucial role in the oxidative stress resistance process. Finally, our study demonstrated that the T6SS1 and siderophore systems in C. pinatubonensis exhibit different responses in combating oxidative stress under low-iron conditions, providing a comprehensive understanding of how bacterial iron acquisition systems function in diverse conditions.IMPORTANCEThe ability to eliminate reactive oxygen species is crucial for bacterial survival. Continuous formation of hydroperoxides can damage metalloenzymes, disrupt DNA integrity, and even result in cell death. While various mechanisms have been identified in other bacterial species to combat oxidative stress, the specific mechanism of oxidative stress resistance in C. pinatubonensis remains unclear. The importance of this study is that we elucidate the mechanism that OxyR-regulated T6SS1 combats oxidative stress by importing iron with the help of bacterial outer membrane vesicle. Moreover, the study highlights the contrasting responses of T6SS1- and siderophore-mediated iron acquisition systems to oxidative stress. This study provides a comprehensive understanding of bacterial iron acquisition and its role in oxidative stress resistance in C. pinatubonensis under low-iron conditions.

Pontibacter toksunensis sp. nov., isolated from soil, and emended descriptions of Pontibacter roseus and Pontibacter akesuensis.

A Gram-reaction-negative, rod-shaped, non-motile, red-pigmented bacterial strain, designated ZLD-7(T), was isolated from a soil sample collected from an arid area in Xinjiang Province in north-west China, and characterized by using a polyphasic taxonomic approach. 16S rRNA gene sequence analysis showed that strain ZLD-7(T) was a member of the genus Pontibacter in the family Cytophagaceae, with sequence similarities of 93.7-96.2 % to type strains of other Pontibacter species. The only isoprenoid quinone of strain ZLD-7(T) was MK-7, and its major cellular fatty acids were summed feature 4 (anteiso-C17 : 1 B and/or iso-C17 : 1 I), iso-C15 : 0, iso-C17 : 0 3-OH and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c). The polar lipid profile consisted of phosphatidylethanolamine, one unidentified aminolipid and four unidentified polar lipids. The DNA G+C content was 47.8 mol%. On the basis of the evidence presented, it is proposed that strain ZLD-7(T) represents a novel species of the genus Pontibacter, for which the name Pontibacter toksunensis sp. nov. is proposed. The type strain is ZLD-7(T) ( = CCTCC AB 208003(T) = KCTC 23984(T)). Emended descriptions of Pontibacter roseus and Pontibacter akesuensis are also proposed.

A Fur-regulated type VI secretion system contributes to oxidative stress resistance and virulence in Yersinia pseudotuberculosis.

The type VI secretion system (T6SS) is a widespread protein secretion apparatus deployed by many Gram-negative bacterial species to interact with competitor bacteria, host organisms, and the environment. Yersinia pseudotuberculosis T6SS4 was recently reported to be involved in manganese acquisition; however, the underlying regulatory mechanism still remains unclear. In this study, we discovered that T6SS4 is regulated by ferric uptake regulator (Fur) in response to manganese ions (Mn2+), and this negative regulation of Fur was proceeded by specifically recognizing the promoter region of T6SS4 in Y. pseudotuberculosis. Furthermore, T6SS4 is induced by low Mn2+ and oxidative stress conditions via Fur, acting as a Mn2+-responsive transcriptional regulator to maintain intracellular manganese homeostasis, which plays important role in the transport of Mn2+ for survival under oxidative stress. Our results provide evidence that T6SS4 can enhance the oxidative stress resistance and virulence for Y. pseudotuberculosis. This study provides new insights into the regulation of T6SS4 via the Mn2+-dependent transcriptional regulator Fur, and expands our knowledge of the regulatory mechanisms and functions of T6SS from Y. pseudotuberculosis.

H3-T6SS of Pseudomonas aeruginosa PA14 contributes to environmental adaptation via secretion of a biofilm-promoting effector.

Microbial species often occur in complex communities and exhibit intricate synergistic and antagonistic interactions. To avoid predation and compete for favorable niches, bacteria have evolved specialized protein secretion systems. The type VI secretion system (T6SS) is a versatile secretion system widely distributed among Gram-negative bacteria that translocates effectors into target cells or the extracellular milieu via various physiological processes. Pseudomonas aeruginosa is an opportunistic pathogen responsible for many diseases, and it has three independent T6SSs (H1-, H2-, and H3-T6SS). In this study, we found that the H3-T6SS of highly virulent P. aeruginosa PA14 is negatively regulated by OxyR and OmpR, which are global regulatory proteins of bacterial oxidative and acid stress. In addition, we identified a H3-T6SS effector PA14_33970, which is located upstream of VgrG3. PA14_33970 interacted directly with VgrG3 and translocated into host cells. Moreover, we found that H3-T6SS and PA14_33970 play crucial roles in oxidative, acid, and osmotic stress resistance, as well as in motility and biofilm formation. PA14_33970 was identified as a new T6SS effector promoting biofilm formation and thus named TepB. Furthermore, we found that TepB contributes to the virulence of P. aeruginosa PA14 toward Caenorhabditis elegans. Overall, our study indicates that H3-T6SS and its biofilm-promoting effector TepB are regulated by OxyR and OmpR, both of which are important for adaptation of P. aeruginosa PA14 to multiple stressors, providing insights into the regulatory mechanisms and roles of T6SSs in P. aeruginosa.

T6SS secretes an LPS-binding effector to recruit OMVs for exploitative competition and horizontal gene transfer.

Outer membrane vesicles (OMVs) can function as nanoscale vectors that mediate bacterial interactions in microbial communities. How bacteria recognize and recruit OMVs inter-specifically remains largely unknown, thus limiting our understanding of the complex physiological and ecological roles of OMVs. Here, we report a ligand-receptor interaction-based OMV recruitment mechanism, consisting of a type VI secretion system (T6SS)-secreted lipopolysaccharide (LPS)-binding effector TeoL and the outer membrane receptors CubA and CstR. We demonstrated that Cupriavidus necator T6SS1 secretes TeoL to preferentially associate with OMVs in the extracellular milieu through interactions with LPS, one of the most abundant components of OMVs. TeoL associated with OMVs can further bind outer membrane receptors CubA and CstR, which tethers OMVs to the recipient cells and allows cargo to be delivered. The LPS-mediated mechanism enables bacterial cells to recruit OMVs derived from different species, and confers advantages to bacterial cells in iron acquisition, interbacterial competition, and horizontal gene transfer (HGT). Moreover, our findings provide multiple new perspectives on T6SS functionality in the context of bacterial competition and HGT, through the recruitment of OMVs.

A secreted effector with a dual role as a toxin and as a transcriptional factor.

Bacteria have evolved multiple secretion systems for delivering effector proteins into the cytosol of neighboring cells, but the roles of many of these effectors remain unknown. Here, we show that Yersinia pseudotuberculosis secretes an effector, CccR, that can act both as a toxin and as a transcriptional factor. The effector is secreted by a type VI secretion system (T6SS) and can enter nearby cells of the same species and other species (such as Escherichia coli) via cell-cell contact and in a contact-independent manner. CccR contains an N-terminal FIC domain and a C-terminal DNA-binding domain. In Y. pseudotuberculosis cells, CccR inhibits its own expression by binding through its DNA-binding domain to the cccR promoter, and affects the expression of other genes through unclear mechanisms. In E. coli cells, the FIC domain of CccR AMPylates the cell division protein FtsZ, inducing cell filamentation and growth arrest. Thus, our results indicate that CccR has a dual role, modulating gene expression in neighboring cells of the same species, and inhibiting the growth of competitors.

Aerobactin-Mediated Iron Acquisition Enhances Biofilm Formation, Oxidative Stress Resistance, and Virulence of Yersinia pseudotuberculosis.

Aerobactin is a citrate-hydroxamate siderophore that is critical for the virulence of pathogenic enteric bacteria. However, although the aerobactin-producing iucABCD-iutA operon is distributed widely in the genomes of Yersinia species, none of the pathogenic Yersinia spp. was found to produce aerobactin. Here, we showed that the iucABCD-iutA operon in the food-borne enteric pathogen Yersinia pseudotuberculosis YPIII is a functional siderophore system involved in iron acquisition. The expression of the operon was found to be directly repressed by the ferric uptake regulator (Fur) in an iron concentration-dependent manner. In addition, we demonstrated that the aerobactin-mediated iron acquisition contributes to bacterial growth under iron-limited conditions. Moreover, we provided evidence that aerobactin plays important roles in biofilm formation, resistance to oxidative stress, ROS removal, and virulence of Y. pseudotuberculosis. Overall, our study not only uncovered a novel strategy of iron acquisition in Y. pseudotuberculosis but also highlighted the importance of aerobactin in the pathogenesis of Y. pseudotuberculosis.

RovM and CsrA Negatively Regulate Urease Expression in Yersinia pseudotuberculosis.

Urease acts as an important acid resistance system and virulence factor that is widespread among microorganisms. RovM is a global regulator that regulates a series of genes and pathways including acid survival systems in the enteric bacterium Yersinia pseudotuberculosis (Yptb). However, whether RovM regulates the urease activity in Yptb was still unknown. In this study, by using qualitative and quantitative urease assays, we show that the urease expression responds to nutrient conditions and the RovM protein represses urease expression by binding to its promoter. A previously reported positive regulator OmpR activates urease activity but RovM plays a dominant role in different nutrient conditions. In addition, carbon storage regulator system A (CsrA), the upstream regulator of RovM, dramatically down-regulates urease activity possibly by its binding to the Shine-Dalgarno (SD) sequence of the mRNA encoding the urease. In conclusion, this study demonstrates that urease activity is strictly controlled by nutrient conditions and is down-regulated by the CsrA-RovM pathway.