The Brucella Cell Envelope.

The cell envelope is a multilayered structure that insulates the interior of bacterial cells from an often chaotic outside world. Common features define the envelope across the bacterial kingdom, but the molecular mechanisms by which cells build and regulate this critical barrier are diverse and reflect the evolutionary histories of bacterial lineages. Intracellular pathogens of the genus Brucella exhibit marked differences in cell envelope structure, regulation, and biogenesis when compared to more commonly studied gram-negative bacteria and therefore provide an excellent comparative model for study of the gram-negative envelope. We review distinct features of the Brucella envelope, highlighting a conserved regulatory system that links cell cycle progression to envelope biogenesis and cell division. We further discuss recently discovered structural features of the Brucella envelope that ensure envelope integrity and that facilitate cell survival in the face of host immune stressors.

The cell division protein FzlA performs a conserved function in diverse alphaproteobacteria.

In almost all bacteria, the tubulin-like GTPase FtsZ polymerizes to form a "Z-ring" that marks the site of division. FtsZ recruits other proteins, collectively known as the divisome, that together remodel and constrict the envelope. Constriction is driven by peptidoglycan (PG) cell wall synthesis by the glycosyltransferase FtsW and the transpeptidase FtsI (FtsWI), but these enzymes require activation to function. How recruitment of FtsZ to the division site leads to FtsWI activation and constriction remains largely unknown. Previous work in our laboratory demonstrated that an FtsZ-binding protein, FzlA, is essential for activation of FtsWI in the alphaproteobacterium Caulobacter crescentus. Additionally, we found that FzlA binds to a DNA translocase called FtsK, suggesting that it may link constriction activation to chromosome segregation. FzlA is conserved throughout Alphaproteobacteria but has only been examined in detail in C. crescentus. Here, we explored whether FzlA function is conserved in diverse Alphaproteobacteria. We assessed FzlA homologs from Rickettsia parkeri and Agrobacterium tumefaciens, and found that, similar to C. crescentus FzlA, they bind directly to FtsZ and localize to midcell. The FtsZ-FzlA interaction interface is conserved, as we demonstrated that FzlA from each of the three species examined can bind to FtsZ from any of the three in vitro. Finally, we determined that A. tumefaciens FzlA can fulfill the essential function of FzlA when produced in C. crescentus, indicating conservation of function. These results suggest that FzlA serves as an important regulator that coordinates chromosome segregation with envelope constriction across diverse Alphaproteobacteria.IMPORTANCECell division is essential for bacterial replication and must be highly regulated to ensure robust remodeling of the cell wall in coordination with segregation of the genome to daughter cells. In Caulobacter crescentus, FzlA plays a major role in regulating this process by activating cell wall synthesis in a manner that couples constriction to chromosome segregation. FzlA is broadly conserved in Alphaproteobacteria, suggesting that it plays a similar function across this class of bacteria. Here, we have shown that, indeed, FzlA biochemical interactions and function are conserved in diverse Alphaproteobacteria. Because FzlA is conserved in Alphaproteobacterial human pathogens, understanding this protein and its interactome could present therapeutic benefits by identifying potential antibiotic targets to treat infections.

The Sinorhizobium meliloti nitrogen-fixing symbiosis requires CbrA-dependent regulation of a DivL and CckA phosphorelay.

The cell cycle is a fundamental process involved in bacterial reproduction and cellular differentiation. For Sinorhizobium meliloti, cell cycle outcomes depend on its growth environment. This bacterium shows a tight coupling of DNA replication initiation with cell division during free-living growth. In contrast, it undergoes a novel program of endoreduplication and terminal differentiation during symbiosis within its host. While several DivK regulators at the top of its CtrA pathway have been shown to play an important role in this differentiation process, there is a lack of resolution regarding the downstream molecular activities required and whether they could be unique to the symbiosis cell cycle. The DivK kinase CbrA is a negative regulator of CtrA activity and is required for successful symbiosis. In this work, spontaneous symbiosis suppressors of ΔcbrA were identified as alleles of divL and cckA. In addition to rescuing symbiotic development, they restore wild-type cell cycle progression to free-living ΔcbrA cells. Biochemical characterization of the S. meliloti hybrid histidine kinase CckA in vitro demonstrates that it has both kinase and phosphatase activities. Specifically, CckA on its own has autophosphorylation activity, and phosphatase activity is induced by the second messenger c-di-GMP. Importantly, the CckAA373S suppressor protein of ΔcbrA has a significant loss in kinase activity, and this is predicted to cause decreased CtrA activity in vivo. These findings deepen our understanding of the CbrA regulatory pathway and open new avenues for further molecular characterization of a network pivotal to the free-living cell cycle and symbiotic differentiation of S. meliloti.IMPORTANCESinorhizobium meliloti is a soil bacterium able to form a nitrogen-fixing symbiosis with certain legumes, including the agriculturally important Medicago sativa. It provides ammonia to plants growing in nitrogen-poor soils and is therefore of agricultural and environmental significance as this symbiosis negates the need for industrial fertilizers. Understanding mechanisms governing symbiotic development is essential to either engineer a more effective symbiosis or extend its potential to non-leguminous crops. Here, we identify mutations within cell cycle regulators and find that they control cell cycle outcomes during both symbiosis and free-living growth. As regulators within the CtrA two-component signal transduction pathway, this study deepens our understanding of a regulatory network shaping host colonization, cell cycle differentiation, and symbiosis in an important model organism.

Evolutionary history influences the microbiomes of a female symbiotic reproductive organ in cephalopods.

Many female squids and cuttlefishes have a symbiotic reproductive organ called the accessory nidamental gland (ANG) that hosts a bacterial consortium involved with egg defense against pathogens and fouling organisms. While the ANG is found in multiple cephalopod families, little is known about the global microbial diversity of these ANG bacterial symbionts. We used 16S rRNA gene community analysis to characterize the ANG microbiome from different cephalopod species and assess the relationship between host and symbiont phylogenies. The ANG microbiome of 11 species of cephalopods from four families (superorder: Decapodiformes) that span seven geographic locations was characterized. Bacteria of class Alphaproteobacteria, Gammaproteobacteria, and Flavobacteriia were found in all species, yet analysis of amplicon sequence variants by multiple distance metrics revealed a significant difference between ANG microbiomes of cephalopod families (weighted/unweighted UniFrac, Bray-Curtis, P = 0.001). Despite being collected from widely disparate geographic locations, members of the family Sepiolidae (bobtail squid) shared many bacterial taxa including (~50%) Opitutae (Verrucomicrobia) and Ruegeria (Alphaproteobacteria) species. Furthermore, we tested for phylosymbiosis and found a positive correlation between host phylogenetic distance and bacterial community dissimilarity (Mantel test r = 0.7). These data suggest that closely related sepiolids select for distinct symbionts from similar bacterial taxa. Overall, the ANGs of different cephalopod species harbor distinct microbiomes and thus offer a diverse symbiont community to explore antimicrobial activity and other functional roles in host fitness.IMPORTANCEMany aquatic organisms recruit microbial symbionts from the environment that provide a variety of functions, including defense from pathogens. Some female cephalopods (squids, bobtail squids, and cuttlefish) have a reproductive organ called the accessory nidamental gland (ANG) that contains a bacterial consortium that protects eggs from pathogens. Despite the wide distribution of these cephalopods, whether they share similar microbiomes is unknown. Here, we studied the microbial diversity of the ANG in 11 species of cephalopods distributed over a broad geographic range and representing 15-120 million years of host divergence. The ANG microbiomes shared some bacterial taxa, but each cephalopod species had unique symbiotic members. Additionally, analysis of host-symbiont phylogenies suggests that the evolutionary histories of the partners have been important in shaping the ANG microbiome. This study advances our knowledge of cephalopod-bacteria relationships and provides a foundation to explore defensive symbionts in other systems.

Novel Alphaproteobacteria transcribe genes for nitric oxide transformation at high levels in a marine oxygen-deficient zone.

Marine oxygen-deficient zones (ODZs) are portions of the ocean where intense nitrogen loss occurs primarily via denitrification and anammox. Despite many decades of study, the identity of the microbes that catalyze nitrogen loss in ODZs is still being elucidated. Intriguingly, high transcription of genes in the same family as the nitric oxide dismutase (nod) gene from Methylomirabilota has been reported in the anoxic core of ODZs. Here, we show that the most abundantly transcribed nod genes in the Eastern Tropical North Pacific ODZ belong to a new order (UBA11136) of Alphaproteobacteria, rather than Methylomirabilota as previously assumed. Gammaproteobacteria and Planctomycetia also transcribe nod, but at lower relative abundance than UBA11136 in the upper ODZ. The nod-transcribing Alphaproteobacteria likely use formaldehyde and formate as a source of electrons for aerobic respiration, with additional electrons possibly from sulfide oxidation. They also transcribe multiheme cytochrome (here named ptd) genes for a putative porin-cytochrome protein complex of unknown function, potentially involved in extracellular electron transfer. Molecular oxygen for aerobic respiration may originate from nitric oxide dismutation via cryptic oxygen cycling. Our results implicate Alphaproteobacteria order UBA11136 as a significant player in marine nitrogen loss and highlight their potential in one-carbon, nitrogen, and sulfur metabolism in ODZs.IMPORTANCEIn marine oxygen-deficient zones (ODZs), microbes transform bioavailable nitrogen to gaseous nitrogen, with nitric oxide as a key intermediate. The Eastern Tropical North Pacific contains the world's largest ODZ, but the identity of the microbes transforming nitric oxide remains unknown. Here, we show that highly transcribed nitric oxide dismutase (nod) genes belong to Alphaproteobacteria of the novel order UBA11136, which lacks cultivated isolates. These Alphaproteobacteria show evidence for aerobic respiration, using oxygen potentially sourced from nitric oxide dismutase, and possess a novel porin-cytochrome protein complex with unknown function. Gammaproteobacteria and Planctomycetia transcribe nod at lower levels. Our results pinpoint the microbes mediating a key step in marine nitrogen loss and reveal an unexpected predicted metabolism for marine Alphaproteobacteria.

Tools for genetic engineering and gene expression control in Novosphingobium aromaticivorans and Rhodobacter sphaeroides.

Alphaproteobacteria have a variety of cellular and metabolic features that provide important insights into biological systems and enable biotechnologies. For example, some species are capable of converting plant biomass into valuable biofuels and bioproducts that have the potential to contribute to the sustainable bioeconomy. Among the Alphaproteobacteria, Novosphingobium aromaticivorans, Rhodobacter sphaeroides, and Zymomonas mobilis show promise as organisms that can be engineered to convert extracted plant lignin or sugars into bioproducts and biofuels. Genetic manipulation of these bacteria is needed to introduce engineered pathways and modulate expression of native genes with the goal of enhancing bioproduct output. Although recent work has expanded the genetic toolkit for Z. mobilis, N. aromaticivorans and R. sphaeroides still need facile, reliable approaches to deliver genetic payloads to the genome and to control gene expression. Here, we expand the platform of genetic tools for N. aromaticivorans and R. sphaeroides to address these issues. We demonstrate that Tn7 transposition is an effective approach for introducing engineered DNA into the chromosome of N. aromaticivorans and R. sphaeroides. We screen a synthetic promoter library to identify isopropyl ß-D-1-thiogalactopyranoside-inducible promoters with regulated activity in both organisms (up to ~15-fold induction in N. aromaticivorans and ~5-fold induction in R. sphaeroides). Combining Tn7 integration with promoters from our library, we establish CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference systems for N. aromaticivorans and R. sphaeroides (up to ~10-fold knockdown in N. aromaticivorans and R. sphaeroides) that can target essential genes and modulate engineered pathways. We anticipate that these systems will greatly facilitate both genetic engineering and gene function discovery efforts in these species and other Alphaproteobacteria.IMPORTANCEIt is important to increase our understanding of the microbial world to improve health, agriculture, the environment, and biotechnology. For example, building a sustainable bioeconomy depends on the efficient conversion of plant material to valuable biofuels and bioproducts by microbes. One limitation in this conversion process is that microbes with otherwise promising properties for conversion are challenging to genetically engineer. Here we report genetic tools for Novosphingobium aromaticivorans and Rhodobacter sphaeroides that add to the burgeoning set of tools available for genome engineering and gene expression in Alphaproteobacteria. Our approaches allow straightforward insertion of engineered pathways into the N. aromaticivorans or R. sphaeroides genome and control of gene expression by inducing genes with synthetic promoters or repressing genes using CRISPR interference. These tools can be used in future work to gain additional insight into these and other Alphaproteobacteria and to aid in optimizing yield of biofuels and bioproducts.

Fontisubflavum oceani gen. nov., sp. nov., isolated from the deep-sea cold seep water of South China Sea.

In this study, we report a Gram-stain-negative, rod-shaped, atrichous and aerobic bacterial strain named CSW1921T, which was isolated from the deep-sea water of a cold seep in South China Sea. Growth of strain CSW1921T occurred at 10.0-35.0 °C (optimum, 30 °C), pH 5.0-10.0 (optimum, pH 8.0-9.0) and with 0-9.0 % (w/v) NaCl (optimum, 1.0-2.0 %). Phylogenetic tree analysis based on 16S rRNA gene sequence or the genomic sequence indicated that strain CSW1921T belonged to the family Rhodobacteraceae and was closely related to Rhodophyticola porphyridii MA-7-27T (97.5 % sequence similarity). Genomic analysis indicated that strain CSW1921T contains a circular chromosome of 3 592 879 bp with G+C content of 60.5 mol%. The predominant respiratory quinone of CSW1921T was ubiquinone-10. The polar lipids of CSW1921T contained phosphatidylglycerol, three unidentified aminolipids, two unidentified phospholipids and two unidentified lipids. The major fatty acids of strain CSW1921T contained C16 : 0, C18 : 1 ω7c 11-methyl and summed feature 8 (C18 : 1 ω7c). The average nucleotide identity, DNA-DNA hybridization and average amino acid identity values between strain CSW1921T and members of its related species were 68.02-69.08 %, 12.7-12.9 % and 46.87-48.08 %, respectively, which were lower than the recommended threshold values for bacterial species or genus delineation. Phylogenetic, physiological, biochemical and morphological analyses suggested that strain CSW1921T represents a novel genus and a novel species of the family Rhodobacteraceae, and the name Fontisubflavum oceani gen. nov., sp. nov. is proposed with the type strain CSW1921T (=MCCC 1K08371T=KCTC 92834T).

Aquibaculum arenosum gen. nov., sp. nov., a novel member of the family Rhodovibrionaceae, isolated from sea sand.

A Gram-negative, non-motile, and creamy-white coloured bacterium, designated CAU 1616T, was isolated from sea sand collected at Ayajin Beach, Goseong-gun, Republic of Korea. The bacterium was found to grow optimally at 37 °C, pH 8.0-8.5, and with 1-5 % (w/v) NaCl. Phylogenetic analyses based on the 16S rRNA gene sequences placed strain CAU 1616T within the order Rhodospirillales. The highest 16S rRNA gene sequence similarity was to Fodinicurvata fenggangensis YIM D812T (94.1 %), Fodinicurvata sediminis YIM D82T (93.7 %), Fodinicurvata halophila BA45ALT (93.6 %) and Algihabitans albus HHTR 118T (92.3 %). Comparing strain CAU 1616T with closely related species (Fodinicurvata fenggangensis YIM D812T and Fodinicurvata sediminis YIM D82T), the average nucleotide identity based on blast+ values were 69.7-69.8 %, the average amino acid identity values were 61.3-61.4 %, and the digital DNA-DNA hybridization values were 18.4-18.5 %. The assembled draft genome of strain CAU 1616T had 29 contigs with an N50 value of 385.8 kbp, a total length of 3 490 371 bp, and a DNA G+C content of 65.1 mol%. The predominant cellular fatty acids were C18 : 1 2-OH, C19 : 0 cyclo ω8c, and summed feature 8 (C18 : 1 ω6c and/or C18 : 1 ω7c). The major respiratory quinone was Q-10. Based on phenotypic, phylogenetic, and chemotaxonomic evidence, strain CAU 1616T represents a novel genus in the family Rhodovibrionaceae, for which the name Aquibaculum arenosum gen. nov., sp. nov. is proposed. The type strain is CAU 1616T (=KCTC 82428T=MCCC 1K06089T).

Parerythrobacter lacustris sp. nov., a novel member of the family Erythrobacteraceae isolated from an inland alpine lake.

A novel bacterium, designated as strain RS5-5T, was isolated from lake water in northwestern China. Cells of the isolate were observed to be rod shaped and Gram stain negative. Its growth occurred at 4-37 â„ƒ, pH 6.5-9.0 and in the presence of 0-5% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain RS5-5T was most closely related to Qipengyuania sediminis GDMCC 1.2497T (97.5%), followed by Erythrobacter dokdonensis DSW-74T (97.3%) and Qipengyuania algicida GDMCC 1.2535T (97.0%). Phylogenomic analysis revealed that strain RS5-5T formed a distinct branch with the genus Parerythrobacter. The sole quinone was ubiquinone-10, and the major fatty acids (≥ 10%) were unsaturated fatty acids including C17:1 ω6c, summed feature 3 (C16:1 ω7c/C16:1 ω6c) and summed feature 8 (C18:1 ω7c/C18:1 ω6c). The polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, one unidentified sphingoglycolipid, three unidentified glycolipids, one unidentified aminoglycolipid, one unidentified aminolipid, two unidentified phospholipids and four unidentified polar lipids. Chemotaxonomic characteristics of strain RS5-5T were coincident with those of the genus Parerythrobacter members. The average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization values between strain RS5-5T and two Parerythrobacter reference strains were in the ranges of 73.2-77.7%, 69.0-78.0% and 18.9-20.4%, respectively. The genomic DNA G + C content of strain RS5-5T was 64.1%. The results of phenotypic, phylogenetic and genomic analyses suggested that strain RS5-5T represents a novel species in the genus Parerythrobacter, for which the name Parerythrobacter lacustris sp. nov. is proposed. The type strain is RS5-5T (= GDMCC 1.3163T = KCTC 92277T).

Novosphingobium album sp. nov., Novosphingobium organovorum sp. nov. and Novosphingobium mangrovi sp. nov. with the organophosphorus pesticides degrading ability isolated from mangrove sediments.

Members of the genus Novosphingobium were frequently isolated from polluted environments and possess great bioremediation potential. Here, three species, designated B2637T, B2580T and B1949T, were isolated from mangrove sediments and might represent novel species in the genus Novosphingobium based on a polyphasic taxonomy study. Phylogenomic analysis revealed that strains B2580T, B1949T and B2637T clustered with Novosphingobium naphthalenivorans NBRC 102051T, 'N. profundi' F72 and N. decolorationis 502str22T, respectively. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between isolates and their closely related species were less than 94 and 54 %, respectively, all below the threshold of species discrimination. The sizes of the genomes of isolates B2580T, B2637T and B1949T ranged from 4.4 to 4.6 Mb, containing 63.3-66.4 % G+C content. Analysis of their genomic sequences identified genes related to pesticide degradation, heavy-metal resistance, nitrogen fixation, antibiotic resistance and sulphur metabolism, revealing the biotechnology potential of these isolates. Except for B2637T, B1949T and B2580T were able to grow in the presence of quinalphos. Results from these polyphasic taxonomic analyses support the affiliation of these strains to three novel species within the genus Novosphingobium, for which we propose the name Novosphingobium album sp. nov. B2580T (=KCTC 72967T=MCCC 1K04555T), Novosphingobium organovorum sp. nov. B1949T (=KCTC 92158T=MCCC 1K03763T) and Novosphingobium mangrovi sp. nov. B2637T (KCTC 72969T=MCCC 1K04460T).

Mariluticola halotolerans gen. nov., sp. nov., a novel member of the family Devosiaceae isolated from South China Sea sediment.

A novel member of class Alphaproteobacteria was isolated from marine sediment of the South China Sea. Cells of strain LMO-2T were Gram-stain negative, greyish in colour, motile, with a single lateral flagellum and short rod in shape with a slight curve. Strain LMO-2T was positive for oxidase and negative for catalase. The bacterium grew aerobically at 10-40 °C (optimum, 25-30 °C), pH 5.5-10.0 (optimum, pH 7.0) and 0-9 % NaCl (w/v; optimum, 2-3 %). Phylogenetic analysis of the 16S rRNA gene sequence and phylogenomic analysis of the whole genome sequence indicated that strain LMO-2T represents a new genus and a new species within the family Devosiaceae, class Alphaproteobacteria, phylum Pseudomonadota. Comparisons of the 16S rRNA gene sequences of strain LMO-2T showed 94.8 % similarity to its closest relative. The genome size is ~3.45 Mbp with a DNA G+C content of 58.17 mol%. The strain possesses potential capability for the degradation of complex organic matter, i.e. fatty acid and benzoate. The predominant cellular fatty acids (>10 %) were C16 : 0 and C18 : 1 ω7c 11-methyl. The sole respiratory quinone was ubiquinone-10. The major identified polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phospholipid. Based on the polyphasic taxonomic data, strain LMO-2T represents a novel genus and a novel species for which the name Mariluticola halotolerans gen. nov., sp. nov., was proposed in the family Devosiaceae. The type strain is LMO-2T (=CGMCC 1.19273T=JCM 34934T).

Maritalea mediterranea sp. nov., isolated from marine plastic residues.

A novel Gram-reaction-negative, aerobic, motile, rod-shaped, grey bacterium, strain P4.10XT, was isolated from plastic debris sampled from shallow waters in the Mediterranean Sea (Valencia, Spain). P4.10XT was catalase- and oxidase-positive, and grew under mesophilic, neutrophilic and halophilic conditions. The 16S rRNA gene sequences revealed that P4.10XT was closely related to Maritalea myrionectae DSM 19524T and Maritalea mobilis E6T (98.25 and 98.03 % sequence similarity, respectively). The DNA G+C content of the genome sequence of P4.10XT was 53.66 %. The genomic indexes average nucleotide identity by blast (ANIb) and digital DNA-DNA hybridization (dDDH) confirmed its classification as representing a novel species of the genus Maritalea. The predominant fatty acids were summed feature 8 (C18 : 1ω7c/C18 : 1ω6c) and C18 : 1 ω7c 11-methyl. The results of this polyphasic study confirm that P4.10XT represents a novel species of the genus Maritalea, for which the name Maritalea mediterranea sp. nov. is proposed (type strain P4.10XT=CECT 30306T = DSM 112386T).

Sandaracinobacteroides sayramensis sp. nov., a yellow-pigmented bacterium isolated from lake water.

A Gram-negative, non-motile, facultatively anaerobic, rod-shaped bacterium, designated strain RS1-74T, was isolated from the surface water of Sayram Lake, Xinjiang Uygur Autonomous Region, China. The strain was able to grow optimally at 30 °C and pH 7.0-7.5, and in the presence of 0-0.5 % (v/w) NaCl. Catalase and oxidase activities were present. H2S was produced. Chemotaxonomic analysis showed Q-10 was the sole respiratory quinone. The polar lipids were composed of phosphatidylethanolamine, diphosphatidylglycerol, two glycolipids, phosphatidylglycerol, sphingoglycolipid and two unidentified lipids. Summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) and summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) were the predominant fatty acids. Phylogenetic analysis based on 16S rRNA gene sequence showed that strain RS1-74T was closely related to 'Sandaracinobacter neustonicus' JCM 30 734 (98.65 %), 'Sandaracinobacter sibiricus' RB16-17 (98.42 %) and Sandaracinobacteroides hominis SZY PN-1T (97.09%). The genomic DNA G+C content was 66.45 mol%. The average nucleotide identity and DNA-DNA hybridization values among the genomes of strain RS1-74T and 'Sandaracinobacter neustonicus' JCM 30734 and Sandaracinobacteroides hominis SZY PN-1T were 78.2 and 77.22 %, and 22.2 and 20.40 %, respectively. Based on the physiological, biochemical, phylogenetic and genomic data, strain RS1-74T represents a novel species within the genus Sandaracinobacteroides, for which the name Sandaracinobacteroides sayramensis sp. nov. is proposed, with type strain RS1-74T (=KCTC 82674T=MCCC 1K06282T).

Expression of toxic genes in Methylorubrum extorquens with a tightly repressed, cumate-inducible promoter.

Methylorubrum extorquens is an important model methylotroph and has enormous potential for the development of C1-based microbial cell factories. During strain construction, regulated promoters with a low background expression level are important genetic tools for expression of potentially toxic genes. Here we present an accordingly optimised promoter, which can be used for that purpose. During construction and testing of terpene production strains harbouring a recombinant mevalonate pathway, strong growth defects were observed which made strain development impossible. After isolation and characterisation of suppressor mutants, we discovered a variant of the cumate-inducible promoter PQ2148 used in this approach. Deletion of 28 nucleotides resulted in an extremely low background expression level, but also reduced the maximal expression strength to about 30% of the original promoter. This tightly repressed promoter version is a powerful module for controlled expression of potentially toxic genes in M. extorquens.

Thetidibacter halocola gen. nov., sp. nov., a novel member within the family Roseobacteraceae isolated from seawater.

A Gram-staining-negative, strictly aerobic, dark beige-colored, rod-shaped, chemoorganoheterotrophic, and catalase- and oxidase-positive bacterium, designated as KMU-90T, was isolated from coastal seawater in the Republic of Korea, and subjected to a polyphasic study. The novel isolate was able to grow at 0-6.0% NaCl concentrations (w/v), pH 6.5-9.5, and 4-45 °C. The 16S rRNA gene sequences-based phylogeny revealed that the novel marine isolate belongs to the family Roseobacteraceae of class Alphaproteobacteria and that it shared the greatest sequence similarity (97.3%) with Aestuariicoccus marinus NAP41T. The novel strain could be distinguished phenotypically from related representatives of the family Roseobacteraceae. The major (> 10%) fatty acids of strain KMU-90T were C18:1 ω7c and C18:1 ω7c 11-methyl and the only respiratory quinone was ubiquinone-10 (Q-10). Strain KMU-90T contained phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, two unidentified aminolipids, an unidentified phospholipid, and three unidentified glycolipids as polar lipids. The assembled draft genome size of strain KMU-90T was 4.84 Mbp with a DNA G + C content of 66.5%. The average nucleotide identity, digital DNA-DNA hybridization, and average amino acid identity values between the genomes of strain KMU-90T and its closely related representatives were 77.0-79.0%, 14.6-20.0%, and 60.0-69.9%, respectively. From the polyphasic taxonomic results obtained, the strain is considered to represent a novel genus and a new species of the family Roseobacteraceae, for which the name Thetidibacter halocola gen. nov., sp. nov. is proposed. The type species is T. halocola, with the type strain KMU-90T (= KCCM 90287T = NBRC 113375T).

The Role of Bacteria-Mitochondria Communication in the Activation of Neuronal Innate Immunity: Implications to Parkinson's Disease.

Mitochondria play a key role in regulating host metabolism, immunity and cellular homeostasis. Remarkably, these organelles are proposed to have evolved from an endosymbiotic association between an alphaproteobacterium and a primitive eukaryotic host cell or an archaeon. This crucial event determined that human cell mitochondria share some features with bacteria, namely cardiolipin, N-formyl peptides, mtDNA and transcription factor A, that can act as mitochondrial-derived damage-associated molecular patterns (DAMPs). The impact of extracellular bacteria on the host act largely through the modulation of mitochondrial activities, and often mitochondria are themselves immunogenic organelles that can trigger protective mechanisms through DAMPs mobilization. In this work, we demonstrate that mesencephalic neurons exposed to an environmental alphaproteobacterium activate innate immunity through toll-like receptor 4 and Nod-like receptor 3. Moreover, we show that mesencephalic neurons increase the expression and aggregation of alpha-synuclein that interacts with mitochondria, leading to their dysfunction. Mitochondrial dynamic alterations also affect mitophagy which favors a positive feedback loop on innate immunity signaling. Our results help to elucidate how bacteria and neuronal mitochondria interact and trigger neuronal damage and neuroinflammation and allow us to discuss the role of bacterial-derived pathogen-associated molecular patterns (PAMPs) in Parkinson's disease etiology.

Salt- and Osmo-Responsive Sensor Histidine Kinases Activate the Bradyrhizobium diazoefficiens General Stress Response to Initiate Functional Symbiosis.

The general stress response (GSR) enables bacteria to sense and overcome a variety of environmental stresses. In alphaproteobacteria, stress-perceiving histidine kinases of the HWE and HisKA_2 families trigger a signaling cascade that leads to phosphorylation of the response regulator PhyR and, consequently, to activation of the GSR σ factor σEcfG. In the nitrogen-fixing bacterium Bradyrhizobium diazoefficiens, PhyR and σEcfG are crucial for tolerance against a variety of stresses under free-living conditions and also for efficient infection of its symbiotic host soybean. However, the molecular players involved in stress perception and activation of the GSR remained largely unknown. In this work, we first showed that a mutant variant of PhyR where the conserved phosphorylatable aspartate residue D194 was replaced by alanine (PhyRD194A) failed to complement the ΔphyR mutant in symbiosis, confirming that PhyR acts as a response regulator. To identify the PhyR-activating kinases in the nitrogen-fixing symbiont, we constructed in-frame deletion mutants lacking single, distinct combinations, or all of the 11 predicted HWE and HisKA_2 kinases, which we named HRXXN histidine kinases HhkA through HhkK. Phenotypic analysis of the mutants and complemented derivatives identified two functionally redundant kinases, HhkA and HhkE, that are required for nodulation competitiveness and during initiation of symbiosis. Using σEcfG-activity reporter strains, we further showed that both HhkA and HhkE activate the GSR in free-living cells exposed to salt and hyperosmotic stress. In conclusion, our data suggest that HhkA and HhkE trigger GSR activation in response to osmotically stressful conditions which B. diazoefficiens encounters during soybean host infection.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

PdeA is required for the rod shape morphology of Brucella abortus.

Cyclic-di-GMP plays crucial role in the cell cycle regulation of the α-Proteobacterium Caulobacter crescentus. Here we investigated its role in the α-Proteobacterium Brucella abortus, a zoonotic intracellular pathogen. Surprisingly, deletion of all predicted cyclic-di-GMP synthesizing or degrading enzymes did not drastically impair the growth of B. abortus, nor its ability to grow inside cell lines. As other Rhizobiales, B. abortus displays unipolar growth from the new cell pole generated by cell division. We found that the phosphodiesterase PdeA, the ortholog of the essential polar growth factor RgsP of the Rhizobiale Sinorhizobium meliloti, is required for rod shape integrity but is not essential for B. abortus growth. Indeed, the radius of the pole is increased by 31 ± 1.7% in a ΔpdeA mutant, generating a coccoid morphology. A mutation in the cyclic-di-GMP phosphodiesterase catalytic site of PdeA does not generate the coccoid morphology and the ΔpdeA mutant kept the ability to recruit markers of new and old poles. However, the presence of PdeA is required in an intra-nasal mouse model of infection. In conclusion, we propose that PdeA contributes to bacterial morphology and virulence in B. abortus, but it is not crucial for polarity and asymmetric growth.

Differential Localization and Functional Specialization of parS Centromere-Like Sites in repABC Replicons of Alphaproteobacteria.

Partitioning systems ensure the stable inheritance of bacterial low-copy-number replicons, such as chromosomes, chromids, and megaplasmids. These loci consist of two genes encoding partition proteins A and B, and at least one parS centromere-like sequence. In chromids and megaplasmids, partitioning systems are often located in the vicinity of replication systems. An extreme example of this co-localization are alphaproteobacterial repABC replicons, where the partition (repAB) and replication (repC) genes form a single operon, with parS sequences usually positioned in close proximity to these genes. In this study, we characterized a more complex repABC system found in Paracoccus aminophilus (Rhodobacterales) megaplasmid pAMI4 (438 kb). Besides the repABC operon with a single parS site, this replicon has a 2-kb non-coding locus positioned 11.5 kb downstream of repC, which contains three additional parS repeats (3parS). We demonstrated that 3parS is bound by partition protein B in vitro and is essential for proper pAMI4 partitioning in vivo. In search of similar loci, we conducted a comparative analysis of parS distribution in other repABC replicons. This revealed different patterns of parS localization in Rhodobacterales and Rhizobiales. However, in both these taxonomic orders, parS sites are almost always located inside or close to the repABC operon. No other 3parS-like loci were found in the closest relatives of pAMI4. Another evolutionarily-independent example of such a locus was identified as a conserved feature in chromosome 2 of Allorhizobium vitis and related replicons. IMPORTANCE The repABC replication/partitioning loci are widespread in extrachromosomal replicons of Alphaproteobacteria. They are evolutionarily diverse, subject to multi-layer self-regulation, and are responsible for the maintenance of different types of replicons, such as plasmids (e.g., Agrobacterium pTi and pRi tumorigenic and rhizogenic plasmids), megaplasmids (e.g., Sinorhizobium pSymA and pSymB) and essential chromids (e.g., secondary chromosomes of Agrobacterium, Brucella and Rhodobacter). In this study, we functionally analyzed an atypical partition-related component of repABC systems, the 3parS locus, found in the P. aminophilus megaplasmid pAMI4. We also identified parS centromere-like site distribution patterns in different groups of repABC replicons and found other unrelated 3parS-like loci, which had been overlooked. Our findings raise questions concerning the biological reasons for differential parS distribution, which may reflect variations in repABC operon regulation as well as different replication and partition modes of replicons belonging to the repABC family.

Arenibacterium arenosum sp. nov., isolated from sea sand.

A Gram-negative, non-motile, short rod-shaped aerobic bacterial strain CAU 1593T was isolated from a coastal sand sample collected in the Republic of Korea. Cells of strain CAU 1593T grew optimally at 30 °C and pH 7.5 in 4% (w/v) NaCl. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain CAU 1593T had the highest similarity to Arenibacterium halophilum (97.5%). The whole genome of strain CAU 1593T was 3,979,826 bp with 26 contigs, and the DNA G + C content was 64.3 mol%. The major fatty acid of strain CAU 1593 T was summed feature 8 (C18:1 ω7c/C18:1 ω6c). Q-10 was the only respiratory quinone. The polar lipids were composed of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, two phosphoglycolipids, an unidentified glycolipid, and an unidentified lipid. Based on the results of chemotaxonomic, phylogenetic, and phenotypic analyses, strain CAU 1593T represents a novel species in the genus Arenibacterium, which is named Arenibacterium arenosum sp. nov. The type strain is CAU 1593T (= KCTC 82402T = MCCC 1K05671T).