A cross-species genome-wide analysis of sequences similar to those involved in DNA uptake bias in the Pastuerellaceae and Neisseriaceae families of pathogenic bacteria.

Acquiring new DNA allows the emergence of drug resistance in bacteria. Some Pasteurellaceae and Neisseriaceae species preferentially take up specific sequence tags. The study of such sequences is therefore relevant. They are over-represented in the genomes of the corresponding species. I found similar sequences to be present only in, but not in all, the genomes of the Pasteurellaceae and Neisseriaceae families. The genomic densities of these sequences are different both between species and between families. Interestingly, the family whose genomes harbor more of such sequences also shows more sequence types. A phylogenetic analysis allowed inferring the possible ancestral Neisseriacean sequence and a nucleotide-by-nucleotide analysis allowed inferring the potential ancestral Pasteurellacean sequence based on its genomic footprint. The method used for this work could be applied to other sequences, including transcription factor binding and repeated DNAs.

Glyphosate perturbs the gut microbiota of honey bees.

Glyphosate, the primary herbicide used globally for weed control, targets the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme in the shikimate pathway found in plants and some microorganisms. Thus, glyphosate may affect bacterial symbionts of animals living near agricultural sites, including pollinators such as bees. The honey bee gut microbiota is dominated by eight bacterial species that promote weight gain and reduce pathogen susceptibility. The gene encoding EPSPS is present in almost all sequenced genomes of bee gut bacteria, indicating that they are potentially susceptible to glyphosate. We demonstrated that the relative and absolute abundances of dominant gut microbiota species are decreased in bees exposed to glyphosate at concentrations documented in the environment. Glyphosate exposure of young workers increased mortality of bees subsequently exposed to the opportunistic pathogen Serratia marcescens Members of the bee gut microbiota varied in susceptibility to glyphosate, largely corresponding to whether they possessed an EPSPS of class I (sensitive to glyphosate) or class II (insensitive to glyphosate). This basis for differences in sensitivity was confirmed using in vitro experiments in which the EPSPS gene from bee gut bacteria was cloned into Escherichia coli All strains of the core bee gut species, Snodgrassella alvi, encode a sensitive class I EPSPS, and reduction in S. alvi levels was a consistent experimental result. However, some S. alvi strains appear to possess an alternative mechanism of glyphosate resistance. Thus, exposure of bees to glyphosate can perturb their beneficial gut microbiota, potentially affecting bee health and their effectiveness as pollinators.

Identification of active denitrifiers by DNA-stable isotope probing and amplicon sequencing reveals Betaproteobacteria as responsible for attenuation of nitrate contamination in a low impacted aquifer.

Groundwater reservoirs constitute important freshwater resources. However, these ecosystems are highly vulnerable to contamination and have to rely on the resident microbiota to attenuate the impact of this contamination. Nitrate is one of the main contaminants found in groundwater, and denitrification is the main process that removes the compound. In this study, the response to nutrient load on indigenous microbial communities in groundwater from a low impacted aquifer in Uruguay was evaluated. Denitrification rates were measured in groundwater samples from three different sites with nitrate, acetate and pyrite amendments. Results showed that denitrification is feasible under in situ nitrate and electron donor concentrations, although the lack of readily available organic energy source would limit the attenuation of higher nitrate concentrations. DNA-stable isotope probing, combined with amplicon sequencing of 16S rRNA, nirS and nirK genes, was used to identify the active denitrifiers. Members of the phylum Betaproteobacteria were the dominant denitrifiers in two of three sites, with different families being observed; members of the genus Vogesella (Neisseriaceae) were key denitrifiers at one site, while the genera Dechloromonas (Rhodocyclaceae) and Comamonas (Comamonadaceae) were the main denitrifiers detected at the other sites.

Formation of indigoidine derived-pigments contributes to the adaptation of Vogesella sp. strain EB to cold aquatic iron-oxidizing environments.

We investigated previously under explored cold aquatic environments of Andean Patagonia, Argentina. Oily sheens similar to an oil spill are frequently observed at the surface of water in creeks and small ponds in these places. Chemical analysis of a water sample revealed the occurrence of high concentrations of iron and the presence of a free insoluble indigoidine-derived pigment. A blue pigment-producing bacterium (strain EB) was isolated from the water sample and identified as Vogesella sp. by molecular analysis. The isolate was able to produce indigoidine and another derived-pigment (here called cryoindigoidine) with strong antifreeze properties. The production of the pigments depended on the cell growth at cold temperatures (below 15 °C), as well as on the attachment of cells to solid surfaces, and iron limitation in the media. The pigments produced by strain EB showed an inhibitory effect on the growth of diverse microorganisms such as Candida albicans, Escherichia coli and Staphylococcus aureus. In addition, pigmented cells were more tolerant to freezing than non-pigmented cells, suggesting a role of cryoindigoidine/indigoidine as a cold-protectant molecule. The possible roles of the pigments in strain EB physiology and its interactions with the iron-rich environment from which the isolate was obtained are discussed. Results of this study suggested an active role of strain EB in the investigated iron-oxidizing ecosystem.

Structural analysis of haemoglobin binding by HpuA from the Neisseriaceae family.

The Neisseriaceae family of bacteria causes a range of diseases including meningitis, septicaemia, gonorrhoea and endocarditis, and extracts haem from haemoglobin as an important iron source within the iron-limited environment of its human host. Herein we report crystal structures of apo- and haemoglobin-bound HpuA, an essential component of this haem import system. The interface involves long loops on the bacterial receptor that present hydrophobic side chains for packing against the surface of haemoglobin. Interestingly, our structural and biochemical analyses of Kingella denitrificans and Neisseria gonorrhoeae HpuA mutants, although validating the interactions observed in the crystal structure, show how Neisseriaceae have the fascinating ability to diversify functional sequences and yet retain the haemoglobin binding function. Our results present the first description of HpuA's role in direct binding of haemoglobin.

Biological redox cycling of iron in nontronite and its potential application in nitrate removal.

Biological redox cycling of structural Fe in phyllosilicates is an important but poorly understood process. The objective of this research was to study microbially mediated redox cycles of Fe in nontronite (NAu-2). During the reduction phase, structural Fe(III) in NAu-2 served as electron acceptor, lactate as electron donor, AQDS as electron shuttle, and dissimilatory Fe(III)-reducing bacterium Shewanella putrefaciens CN32 as mediator in bicarbonate- and PIPES-buffered media. During the oxidation phase, biogenic Fe(II) served as electron donor and nitrate as electron acceptor. Nitrate-dependent Fe(II)-oxidizing bacterium Pseudogulbenkiania sp. strain 2002 was added as mediator in the same media. For all three cycles, structural Fe in NAu-2 was able to reversibly undergo three redox cycles without significant dissolution. Fe(II) in bioreduced samples occurred in two distinct environments, at edges and in the interior of the NAu-2 structure. Nitrate reduction to nitrogen gas was coupled with oxidation of edge-Fe(II) and part of interior-Fe(II) under both buffer conditions, and its extent and rate did not change with Fe redox cycles. These results suggest that biological redox cycling of structural Fe in phyllosilicates is a reversible process and has important implications for biogeochemical cycles of carbon, nitrogen, and other nutrients in natural environments.

Ligand-enhanced abiotic iron oxidation and the effects of chemical versus biological iron cycling in anoxic environments.

This study introduces a newly isolated, genetically tractable bacterium ( Pseudogulbenkiania sp. strain MAI-1) and explores the extent to which its nitrate-dependent iron-oxidation activity is directly biologically catalyzed. Specifically, we focused on the role of iron chelating ligands in promoting chemical oxidation of Fe(II) by nitrite under anoxic conditions. Strong organic ligands such as nitrilotriacetate and citrate can substantially enhance chemical oxidation of Fe(II) by nitrite at circumneutral pH. We show that strain MAI-1 exhibits unambiguous biological Fe(II) oxidation despite a significant contribution (∼30-35%) from ligand-enhanced chemical oxidation. Our work with the model denitrifying strain Paracoccus denitrificans further shows that ligand-enhanced chemical oxidation of Fe(II) by microbially produced nitrite can be an important general side effect of biological denitrification. Our assessment of reaction rates derived from literature reports of anaerobic Fe(II) oxidation, both chemical and biological, highlights the potential competition and likely co-occurrence of chemical Fe(II) oxidation (mediated by microbial production of nitrite) and truly biological Fe(II) oxidation.

Identification of active denitrifiers in rice paddy soil by DNA- and RNA-based analyses.

Denitrification occurs markedly in rice paddy fields; however, few microbes that are actively involved in denitrification in these environments have been identified. In this study, we used a laboratory soil microcosm system in which denitrification activity was enhanced. DNA and RNA were extracted from soil at six time points after enhancing denitrification activity, and quantitative PCR and clone library analyses were performed targeting the 16S rRNA gene and denitrification functional genes (nirS, nirK and nosZ) to clarify which microbes are actively involved in denitrification in rice paddy soil. Based on the quantitative PCR results, transcription levels of the functional genes agreed with the denitrification activity, although gene abundance did not change at the DNA level. Diverse denitrifiers were detected in clone library analysis, but comparative analysis suggested that only some of the putative denitrifiers, especially those belonging to the orders Neisseriales, Rhodocyclales and Burkholderiales, were actively involved in denitrification in rice paddy soil.

Draft genome sequence of the anaerobic, nitrate-dependent, Fe(II)-oxidizing bacterium Pseudogulbenkiania ferrooxidans strain 2002.

Pseudogulbenkiania ferrooxidans strain 2002 was isolated as a lithoautotrophic, Fe(II)-oxidizing, nitrate-reducing bacterium. Phylogenetically, it is in a clade within the family Neisseriaceae in the order Nessieriales of the class Betaproteobacteria. It is anticipated that comparative genomic analysis of this strain with other nitrate-dependent, Fe(II)-oxidizing bacteria will aid in the elucidation of the genetics and biochemistry underlying this critically important geochemical metabolism.

Sequence-based predictions of lipooligosaccharide diversity in the Neisseriaceae and their implication in pathogenicity.

Endotoxin [Lipopolysaccharide (LPS)/Lipooligosaccharide (LOS)] is an important virulence determinant in gram negative bacteria. While the genetic basis of endotoxin production and its role in disease in the pathogenic Neisseria has been extensively studied, little research has focused on the genetic basis of LOS biosynthesis in commensal Neisseria. We determined the genomic sequences of a variety of commensal Neisseria strains, and compared these sequences, along with other genomic sequences available from various sequencing centers from commensal and pathogenic strains, to identify genes involved in LOS biosynthesis. This allowed us to make structural predictions as to differences in LOS seen between commensal and pathogenic strains. We determined that all neisserial strains possess a conserved set of genes needed to make a common 3-Deoxy-D-manno-octulosonic acid -heptose core structure. However, significant genomic differences in glycosyl transferase genes support the published literature indicating compositional differences in the terminal oligosaccharides. This was most pronounced in commensal strains that were distally related to the gonococcus and meningococcus. These strains possessed a homolog of heptosyltransferase III, suggesting that they differ from the pathogenic strains by the presence a third heptose. Furthermore, most commensal strains possess homologs of genes needed to synthesize lipopolysaccharide (LPS). N. cinerea, a commensal species that is highly related to the gonococcus has lost the ability to make sialyltransferase. Overall genomic comparisons of various neisserial strains indicate that significant recombination/genetic acquisition/loss has occurred within the genus, and this muddles proper speciation.

Chitinolyticbacter meiyuanensis SYBC-H1T, gen. nov., sp. nov., a chitin-degrading bacterium isolated from soil.

A novel aerobic mesophilic bacterial strain SYBC-H1(T) capable of degrading chitin was isolated and classified in this study. The strain exhibited strong chitinolytic activity and was a Gram-negative, curved, rod-shaped, and motile bacterium. Growth of this strain was observed between 10 and 41°C and between pH 3.5 and 9.5. The DNA G + C content of strain SYBC-H1(T) was 53.25 mol%. The cellular fatty acids (>5%) were 12:0 iso 3-OH (5.87%), 16:0 (28.16%), and 18:1ω7c (20.48%). Phylogenetic analysis based on 16S rRNA gene sequence similarity revealed that strain SYBC-H1(T) belonged to the family Neisseriaceae, and was distantly related (95.0% similarity) to the genus Chitiniphilus. Its phenotype was unique and genetic and phylogenetic analysis experiments suggested that strain SYBC-H1(T) represented the type strain (CGMCC 3438(T), ATCC BAA-2140(T)) of a novel genus, for which the name Chitinolyticbacter meiyuanensis SYBC-H1(T) gen. nov., sp. nov. was proposed. The highest enzymatic activity of chitinase (9.6 U/ml) was obtained at 72 h in 250 ml shake flasks. The 16S rRNA gene sequence of SYBC-H1(T) has been deposited in GenBank under the accession number GQ981314.

Deefgea chitinilytica sp. nov., isolated from a wetland.

A bacterial strain, designated Nsw-4(T), was isolated from a water sample of Niao-Song Wetland Park in Taiwan and was characterized by using a polyphasic taxonomic approach. Strain Nsw-4(T) was Gram-negative, aerobic, ivory-coloured, rod-shaped and motile by means of a polar flagellum. Growth occurred at 15-37 degrees C, pH 6.0-8.0 and 0-2 % NaCl. Phylogenetic analyses based on 16S rRNA gene sequences showed that the strain belonged to the genus Deefgea and that its closest neighbour was Deefgea rivuli WB 3.4-79(T) (96.9 %). The results of physiological and biochemical tests allowed the clear phenotypic differentiation of this isolate from D. rivuli WB 3.4-79(T). The major fatty acids were C16 : 1omega7c and C16 : 0. The G+C content of the genomic DNA was 53.7 mol%. On the basis of 16S rRNA gene sequence analysis and the chemotaxonomic and physiological data, strain Nsw-4(T) should be classified as representing a novel species and the second member of the genus Deefgea, for which the name Deefgea chitinilytica sp. nov. is proposed. The type strain is Nsw-4(T) (=BCRC 17934(T)=LMG 24817(T)).

Chitinibacter alvei sp. nov., isolated from stream water.

A Gram-negative, aerobic bacterium, designated strain TNR-14T, was isolated from water of a stream located in southern Taiwan. Cells were rod-shaped and motile by means of a polar flagellum. The isolate exhibited optimum growth at 20 degrees C, at pH 7.0 and in the absence of NaCl. Phylogenetic analyses based on 16S rRNA gene sequences showed that the strain belonged to the genus Chitinibacter and was related most closely to the type strain of Chitinibacter tainanensis (96.0% similarity). The predominant cellular fatty acids were summed feature 3 (C16:1omega7c and/or C16:1omega6c; 54.03%) and C16:0 (27.22%). The DNA G+C content of strain TNR-14T was 57.5 mol%. The results of physiological and biochemical tests and chemotaxonomic data allowed the clear phenotypic differentiation of the new isolate from Chitinibacter tainanensis. Strain TNR-14T is therefore considered to represent a novel species of the genus Chitinibacter, for which the name Chitinibacter alvei sp. nov. is proposed. The type strain is TNR-14T (=LMG 25206T =DSM 22217T =BCRC 17968T).

Vogesella mureinivorans sp. nov., a peptidoglycan-degrading bacterium from lake water.

A novel, non-pigmented, rod-shaped, Gram-negative strain was isolated from mesotrophic lake water in Zealand, Denmark. Phylogenetic analysis of the 16S rRNA gene sequence of the bacterium, designated strain 389(T), indicated that the strain belonged to the genus Vogesella and formed a monophyletic group with Vogesella perlucida DS-28(T) (99.1 % nucleotide similarity); it was less related to Vogesella indigofera ATCC 19706(T) (96.9 % similarity) and Vogesella lacus LMG 24504(T) (96.8 % similarity). Hybridization of DNA from strain 389(T) and V. perlucida demonstrated a reassociation of 50.6 ± 9.6 %. The DNA G+C content of strain 389(T) was 61.2 mol%. The fatty acid profile of the strain differed from those of the other strains representing the genus Vogesella by a high content of C16:1ω7c and/or iso-C15:0 2-OH (71.6 %) and a lower content of C16: 0. Strain 389(T) was capable of degrading peptidoglycan and had chitinase and lysozyme activities, possibly associated with the degradation of peptidoglycan, and had capacity for degradation of several other polymer compounds. Based on phenotypic and genotypic characteristics, strain 389(T) represents a novel species, for which we propose the name Vogesella mureinivorans sp. nov. The type strain is 389(T) (=DSM 21247(T) =LMG 25302(T)).

Silvimonas iriomotensis sp. nov. and Silvimonas amylolytica sp. nov., new members of the class Betaproteobacteria isolated from the subtropical zone in Japan.

The taxonomic positions of two bacterial strains, ir6-1(T) and ir6-4(T), isolated from soil collected in Iriomote Island in Japan, were determined by using a polyphasic taxonomic approach. The strains were facultatively anaerobic, motile and Gram-stain-negative rods and their optimum pH for growth was pH 4.0. Their major respiratory quinone was ubiquinone-8 and the predominant cellular fatty acids were C(14 : 0), C(16 : 0), C(16 : 1)omega7c and C(18 : 1)omega7c. The G+C content of the genomic DNA of ir6-1(T) and ir6-4(T) was 59.9 and 57.5 mol%, respectively. Phylogenetic analysis based on 16S rRNA gene sequences showed that the strains clustered with the genus Silvimonas in the class Betaproteobacteria. DNA-DNA similarities were lower than 53 % among ir6-1(T), ir6-4(T) and Silvimonas terrae NBRC 100961(T), and these strains could be differentiated from each other by several phenotypic characters. Based on these results, we propose the emendation of the genus Silvimonas and inclusion of two novel species, Silvimonas iriomotensis sp. nov. (type strain ir6-1(T)=NBRC 103188(T) =CGMCC 1.8859(T) =KCTC 22513(T)) and Silvimonas amylolytica sp. nov. (ir6-4(T) =NBRC 103189(T) =CGMCC 1.8860(T) =KCTC 22514(T)).

Andreprevotia lacus sp. nov., isolated from a fish-culture pond.

A bacterial strain designated GFC-1(T) was isolated from a fish-culture pond in Taiwan and was characterized by using the polyphasic taxonomic approach. Strain GFC-1(T) was Gram-negative, aerobic, rod-shaped, motile and non-spore-forming. Phylogenetic analyses based on 16S rRNA gene sequences showed that the strain belonged to the genus Andreprevotia of the family Neisseriaceae and its closest neighbour was Andreprevotia chitinilytica JS11-7(T) (97.0 % sequence similarity). The results of physiological and biochemical tests allowed clear phenotypic differentiation of isolate GFC-1(T) from A. chitinilytica JS11-7(T). The major fatty acids were summed feature 3 (C(16 : 1)omega7c and/or iso-C(15 : 0) 2-OH), C(16 : 0) and C(18 : 1)omega7c. The DNA G+C content was 63.0 mol%. On the basis of 16S rRNA gene sequence analysis and chemotaxonomic and physiological data, strain GFC-1(T) should be classified as representing a novel species and a second member of the genus Andreprevotia, for which the name Andreprevotia lacus sp. nov. is proposed. The type strain is GFC-1(T) (=BCRC 17832(T)=LMG 24502(T)).

Chitiniphilus shinanonensis gen. nov., sp. nov., a novel chitin-degrading bacterium belonging to Betaproteobacteria.

A bacterial strain capable of degrading chitin, strain SAY3T, was isolated from moat water of Ueda Castle in Nagano Prefecture, Japan. The strain was gram-negative, curved rod-shaped, facultatively anaerobic, and motile with a single polar flagellum. It grew well with chitin as a sole carbon source. The cellular fatty acids profiles showed the presence of C16:1 omega7c and C16:0 as the major components. The G+C content of DNA was 67.6 mol% and Q-8 was the major respiratory quinone. A 16S rRNA gene sequence-based phylogenetic analysis showed the strain belonged to the family Neisseriaceae but was distantly related (94% identity) to any previously known species. Since the strain was clearly distinct from closely related genera in phenotypic and chemotaxonomic characteristics, it should be classified under a new genus and a new species. We propose the name Chitiniphilus shinanonensis gen. nov., sp. nov. The type strain is SAY3T (=NBRC 104970T=NICMB 14509T).

An intragenic distribution bias of DNA uptake sequences in Pasteurellaceae and Neisseriae.

Most sequenced strains from Pasteurellaceae and Neisseriae contain hundreds to thousands of uptake sequence (US) motifs in their genome, which are associated with natural competence for DNA uptake. The mechanism of their recognition is still unclear, and I searched for intragenic location patterns of these motifs for clues about their distribution. In all cases, one orientation of the US has a higher occurrence in the reading frame, and in all Pasteurellaceae, the US and the reverse complement motifs are biased towards the gene termini. These findings could help design experimental set-ups to study preferential DNA uptake, thereby further unravelling the phenomenon of natural competence.

Silvimonas terrae gen. nov., sp. nov., a novel chitin-degrading facultative anaerobe belonging to the 'Betaproteobacteria'.

A taxonomic study was carried out on a bacterial strain, designated KM-45T, isolated from forest soil collected near Daejeon, South Korea. Comparative 16S rRNA gene sequence analysis indicated a clear affiliation of this bacterium to the 'Betaproteobacteria' and that it was related most closely to Chitinibacter tainanensis BCRC 17254T, Formivibrio citricus DSM 6150T and Iodobacter fluviatilis ATCC 33051T (92.4, 91.2 and 88.9 % 16S rRNA gene sequence similarity, respectively). Cells were Gram-negative, facultatively anaerobic, motile and rod-shaped. The strain grew well on R2A medium and utilized a broad spectrum of carbon sources. The G+C content of the genomic DNA was 58 mol% and the predominant ubiquinone was Q-8. Major fatty acids were C(16 : 0), C(16 : 1)omega7c/iso-C(15 : 0) 2-OH, C(18 : 1)omega7c/omega9t/omega12t and C(17 : 0) cyclo. On the basis of the evidence presented, it is proposed that strain KM-45T should be placed in a novel genus and species, for which the name Silvimonas terrae gen. nov., sp. nov. is proposed. The type strain is KM-45T (=KCTC 12358T=NBRC 100961T).