Vibrio syngnathi sp. nov., a fish pathogen, isolated from the Kiel Fjord.

A new Vibrio strain, K08M4T, was isolated from the broad-nosed pipefish Syngnathus typhle in the Kiel Fjord. Infection experiments revealed that K08M4T was highly virulent for juvenile pipefish. Cells of strain K08M4T were Gram-stain-negative, curved rod-shaped and motile by means of a single polar flagellum. The strain grew aerobically at 9-40° C, at pH 4-10.5 and it tolerated up to 12 % (w/v) NaCl. The most prevalent (>10 %) cellular fatty acids of K08M4T were C16 : 1 ω7c and C16 : 0. Whole-genome comparisons revealed that K08M4T represents a separate evolutionary lineage that is distinct from other Vibrio species and falls within the Splendidus clade. The genome is 4,886,292 bp in size, consists of two circular chromosomes (3,298,328 and 1, 587,964 bp) and comprises 4,178 protein-coding genes and 175 RNA genes. In this study, we describe the phenotypic features of the new isolate and present the annotation and analysis of its complete genome sequence. Based on these data, the new isolate represents a new species for which we propose the name Vibrio syngnathi sp. nov. The type strain is K08M4T (=DSM 109818T=CECT 30086T).

EslB Is Required for Cell Wall Biosynthesis and Modification in Listeria monocytogenes.

Lysozyme is an important component of the innate immune system. It functions by hydrolyzing the peptidoglycan (PG) layer of bacteria. The human pathogen Listeria monocytogenes is intrinsically lysozyme resistant. The peptidoglycan N-deacetylase PgdA and O-acetyltransferase OatA are two known factors contributing to its lysozyme resistance. Furthermore, it was shown that the absence of components of an ABC transporter, referred to here as EslABC, leads to reduced lysozyme resistance. How its activity is linked to lysozyme resistance is still unknown. To investigate this further, a strain with a deletion in eslB, coding for a membrane component of the ABC transporter, was constructed in L. monocytogenes strain 10403S. The eslB mutant showed a 40-fold reduction in the MIC to lysozyme. Analysis of the PG structure revealed that the eslB mutant produced PG with reduced levels of O-acetylation. Using growth and autolysis assays, we showed that the absence of EslB manifests in a growth defect in media containing high concentrations of sugars and increased endogenous cell lysis. A thinner PG layer produced by the eslB mutant under these growth conditions might explain these phenotypes. Furthermore, the eslB mutant had a noticeable cell division defect and formed elongated cells. Microscopy analysis revealed that an early cell division protein still localized in the eslB mutant, indicating that a downstream process is perturbed. Based on our results, we hypothesize that EslB affects the biosynthesis and modification of the cell wall in L. monocytogenes and is thus important for the maintenance of cell wall integrity.IMPORTANCE The ABC transporter EslABC is associated with the intrinsic lysozyme resistance of Listeria monocytogenes However, the exact role of the transporter in this process and in the physiology of L. monocytogenes is unknown. Using different assays to characterize an eslB deletion strain, we found that the absence of EslB affects not only lysozyme resistance but also endogenous cell lysis, cell wall biosynthesis, cell division, and the ability of the bacterium to grow in media containing high concentrations of sugars. Our results indicate that EslB is, by means of a yet-unknown mechanism, an important determinant for cell wall integrity in L. monocytogenes.

Phage vB_BveM-Goe7 represents a new genus in the subfamily Bastillevirinae.

Bacillus velezensis FZB42 is a Gram-positive, endospore-forming rhizobacterium that is associated with plant roots and promotes plant growth. It was used as host to isolate phage vB_BveM-Goe7 (Goe7). Goe7 exhibits a Myoviridae morphology with a contractile tail and an icosahedral head. Its genome is 158,674 bp in size and contains 5137-bp-long terminal repeats (LTRs). It also contains five tRNA-encoding genes and 251 coding DNA sequences (CDS), of which 65 were annotated. The adsorption constant of Goe7 is 6.1 ± 0.24 × 10-8 ml/min, with a latency period of 75 min and a burst size of 114 particles per burst. A BLASTn sequence comparison against the non-redundant nucleotide database of NCBI revealed that Goe7 is most similar to Bacillus subtilis phage vB_BsuM-Goe3.

Phage vB_BmeM-Goe8 infecting Bacillus megaterium DSM319.

vB_BmeM-Goe8 is a phage preying on Bacillus megaterium. Its genome has a GC content of 38.9%, is 161,583 bp in size, and has defined ends consisting of 7436-bp-long terminal repeats. It harbours 11 genes encoding tRNAs and 246 coding DNA sequences, 66 of which were annotated. The particle reveals Myoviridae morphology, and the formation of a double baseplate upon tail sheath contraction indicates morphological relatedness to the group of SPO1-like phages. BLASTn comparison against the NCBI non-redundant nucleotide database revealed that Bacillus phage Mater is the closest relative of vB_BmeM-Goe8.

Complete genome sequence of the virus isolate vB_BthM-Goe5 infecting Bacillus thuringiensis.

Bacillus thuringiensis (Bt) is non-pathogenic for humans and serves as a biological control agent in agriculture. Understanding its phages will help to prevent industrial production loss of Bt products and will lead to a better understanding of phages in general. The complete genome of the new B. thuringiensis phage isolate vB_BthM-Goe5 (Goe5) was sequenced, revealing a linear 157,804-bp-long dsDNA chromosome flanked by 2579-bp-long terminal repeats. It contains two tRNAs and 272 protein coding regions, 69 of which could be assigned with an annotation. Morphological investigation, using transmission electron microscopy, revealed Myoviridae morphology. The formation of a double baseplate upon tail sheath contraction indicates a link to the group of SPO1-related phages. Comparative genomics with all Bacillus-related viral genomes available in the NCBI genome database during this investigation indicated that Goe5 was a unique isolate, with Bacillus phage Bastille as its closest relative.

Description of 'Candidatus Methylocucumis oryzae', a novel Type I methanotroph with large cells and pale pink colour, isolated from an Indian rice field.

An elliptical to cucumber-shaped methanotroph with large cells was isolated from a rice rhizosphere in Western India. Strain Sn10-6 is one of the first methanotrophs to be isolated from Indian rice fields. Cells of Sn10-6 are Gram-negative, motile, large in size (3-6 µm × 1.5-2 µm) and contain intracellular cytoplasmic membrane stacks. Colonies of Sn10-6 and liquid cultures have a pale pink colour. Strain Sn10-6 was initially isolated under micro-oxic conditions but later adapted to grow under fully oxic conditions. The major fatty acids present were identified as C16:1ω6c, C16:1ω7c and C16:0 and ubiquinone was found to be the major quinone. The 16S rRNA gene sequence of strain Sn10-6 displays high similarity to the genes of Methylovulum psychrotolerans Sph1T (93.6%) followed by Methylosarcina fibrata AML-C10T (93.5%) and about 90-93% similarity to the genes of known species of Type I methanotrophic genera from the family Methylococcaceae. The draft genome information indicated that the G + C content of strain Sn10-6 is 43.9 mol%. Phylogenetic trees using 16S rRNA gene and the particulate methane mono-oxygenase sequences unequivocally placed Sn10-6 close to the genus Methylovulum. Based on the 16S rRNA gene differences, morphological characters and draft genome information, strain Sn10-6 (=MCC 3492 =KCTC 15683) is described here as the type strain of a novel species within a new genus, 'Candidatus Methylocucumis oryzae'.

Cyclic Di-GMP Regulates Type IV Pilus-Dependent Motility in Myxococcus xanthus.

UNLABELLED: The nucleotide-based second messenger bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) is involved in regulating a plethora of processes in bacteria that are typically associated with lifestyle changes. Myxococcus xanthus undergoes major lifestyle changes in response to nutrient availability, with the formation of spreading colonies in the presence of nutrients and spore-filled fruiting bodies in the absence of nutrients. Here, we investigated the function of c-di-GMP in M. xanthus and show that this bacterium synthesizes c-di-GMP during growth. Manipulation of the c-di-GMP level by expression of either an active, heterologous diguanylate cyclase or an active, heterologous phosphodiesterase correlated with defects in type IV pilus (T4P)-dependent motility, whereas gliding motility was unaffected. An increased level of c-di-GMP correlated with reduced transcription of the pilA gene (which encodes the major pilin of T4P), reduced the assembly of T4P, and altered cell agglutination, whereas a decreased c-di-GMP level correlated with altered cell agglutination. The systematic inactivation of the 24 genes in M. xanthus encoding proteins containing GGDEF, EAL, or HD-GYP domains, which are associated with c-di-GMP synthesis, degradation, or binding, identified three genes encoding proteins important for T4P-dependent motility, whereas all mutants had normal gliding motility. Purified DmxA had diguanylate cyclase activity, whereas the hybrid histidine protein kinases TmoK and SgmT, each of which contains a GGDEF domain, did not have diguanylate cyclase activity. These results demonstrate that c-di-GMP is important for T4P-dependent motility in M. xanthus. IMPORTANCE: We provide the first direct evidence that M. xanthus synthesizes c-di-GMP and demonstrate that c-di-GMP is important for T4P-dependent motility, whereas we did not obtain evidence that c-di-GMP regulates gliding motility. The data presented uncovered a novel mechanism for regulation of T4P-dependent motility, in which increased levels of c-di-GMP inhibit transcription of the pilA gene (which encodes the major pilin of T4P), ultimately resulting in the reduced assembly of T4P. Moreover, we identified an enzymatically active diguanylate cyclase that is important for T4P-dependent motility.

Biodiversity of green algae covering artificial hard substrate surfaces in a suburban environment: a case study using molecular approaches.

In Middle European suburban environments green algae often cover open surfaces of artificial hard substrates. Microscopy reveals the Apatococcus/Desmococcus morphotype predominant over smaller coccoid forms. Adverse conditions such as limited water availability connected with high PAR and UV irradiance may narrow the algal diversity to a few specialists in these subaerial habitats. We used rRNA gene cloning/sequencing from both DNA extracts of the biofilms without culturing as well as cultures, for the unambiguous determination of the algal composition and to assess the algal diversity more comprehensively. The culture independent approach revealed mainly just two genera (Apatococcus, Trebouxia) for all study sites and five molecular operational taxonomic units (OTUs) for a particular study site, which based on microscopic observation was the one with the highest morphological diversity. The culture approach, however, revealed seven additional OTUs from five genera (Chloroidium, Coccomyxa, Coenochloris, Pabia, Klebsormidium) and an unidentified trebouxiophyte lineage for that same site; only two OTUs were shared by both approaches. Two OTUs or species were recovered for which references have been isolated only from Antarctica so far. However, the internal transcribed spacer (ITS) sequence differences among them supported they are representing distinct populations of the same species. Within Apatococcus five clearly distinct groups of ITS sequences, each putatively representing a distinct species, were recovered with three or four such ITS types co-occurring at the same study site. Except for the streptophyte Klebsormidium only members of Trebouxiophyceae were detected suggesting these algae may be particularly well-adapted to subaerial habitats.

PomZ, a ParA-like protein, regulates Z-ring formation and cell division in Myxococcus xanthus.

Accurate positioning of the division site is essential to generate appropriately sized daughter cells with the correct chromosome number. In bacteria, division generally depends on assembly of the tubulin homologue FtsZ into the Z-ring at the division site. Here, we show that lack of the ParA-like protein PomZ in Myxococcus xanthus resulted in division defects with the formation of chromosome-free minicells and filamentous cells. Lack of PomZ also caused reduced formation of Z-rings and incorrect positioning of the few Z-rings formed. PomZ localization is cell cycle regulated, and PomZ accumulates at the division site at midcell after chromosome segregation but prior to FtsZ as well as in the absence of FtsZ. FtsZ displayed cooperative GTP hydrolysis in vitro but did not form detectable filaments in vitro. PomZ interacted with FtsZ in M. xanthus cell extracts. These data show that PomZ is important for Z-ring formation and is a spatial regulator of Z-ring formation and cell division. The cell cycle-dependent localization of PomZ at midcell provides a mechanism for coupling cell cycle progression and Z-ring formation. Moreover, the data suggest that PomZ is part of a system that recruits FtsZ to midcell, thereby, restricting Z-ring formation to this position.

The orphan histidine protein kinase SgmT is a c-di-GMP receptor and regulates composition of the extracellular matrix together with the orphan DNA binding response regulator DigR in Myxococcus xanthus.

In Myxococcus xanthus the extracellular matrix is essential for type IV pili-dependent motility and starvation-induced fruiting body formation. Proteins of two-component systems including the orphan DNA binding response regulator DigR are essential in regulating the composition of the extracellular matrix. We identify the orphan hybrid histidine kinase SgmT as the partner kinase of DigR. In addition to kinase and receiver domains, SgmT consists of an N-terminal GAF domain and a C-terminal GGDEF domain. The GAF domain is the primary sensor domain. The GGDEF domain binds the second messenger bis-(3'-5')-cyclic-dimeric-GMP (c-di-GMP) and functions as a c-di-GMP receptor to spatially sequester SgmT. We identify the DigR binding site in the promoter of the fibA gene, which encodes an abundant extracellular matrix metalloprotease. Whole-genome expression profiling experiments in combination with the identified DigR binding site allowed the identification of the DigR regulon and suggests that SgmT/DigR regulates the expression of genes for secreted proteins and enzymes involved in secondary metabolite synthesis. We suggest that SgmT/DigR regulates extracellular matrix composition and that SgmT activity is regulated by two sensor domains with ligand binding to the GAF domain resulting in SgmT activation and c-di-GMP binding to the GGDEF domain resulting in spatial sequestration of SgmT.

Localization of Methyl-Coenzyme M reductase as metabolic marker for diverse methanogenic Archaea.

Methyl-Coenzyme M reductase (MCR) as key enzyme for methanogenesis as well as for anaerobic oxidation of methane represents an important metabolic marker for both processes in microbial biofilms. Here, the potential of MCR-specific polyclonal antibodies as metabolic marker in various methanogenic Archaea is shown. For standard growth conditions in laboratory culture, the cytoplasmic localization of the enzyme in Methanothermobacter marburgensis, Methanothermobacter wolfei, Methanococcus maripaludis, Methanosarcina mazei, and in anaerobically methane-oxidizing biofilms is demonstrated. Under growth limiting conditions on nickel-depleted media, at low linear growth of cultures, a fraction of 50-70% of the enzyme was localized close to the cytoplasmic membrane, which implies "facultative" membrane association of the enzyme. This feature may be also useful for assessment of growth-limiting conditions in microbial biofilms.

Deposition of biogenic iron minerals in a methane oxidizing microbial mat.

The syntrophic community between anaerobic methanotrophic archaea and sulfate reducing bacteria forms thick, black layers within multi-layered microbial mats in chimney-like carbonate concretions of methane seeps located in the Black Sea Crimean shelf. The microbial consortium conducts anaerobic oxidation of methane, which leads to the formation of mainly two biomineral by-products, calcium carbonates and iron sulfides, building up these chimneys. Iron sulfides are generated by the microbial reduction of oxidized sulfur compounds in the microbial mats. Here we show that sulfate reducing bacteria deposit biogenic iron sulfides extra- and intracellularly, the latter in magnetosome-like chains. These chains appear to be stable after cell lysis and tend to attach to cell debris within the microbial mat. The particles may be important nuclei for larger iron sulfide mineral aggregates.

Brevundimonas and Serratia as host systems for assessing associated environmental viromes and phage diversity by complementary approaches.

Focusing on visible plaques for phage isolation leaves the question if we miss the diversity of non-plaque forming phages. We addressed this question through direct plaque-based isolation by employing the new hosts Brevundimonas pondensis LVF1 and Serratia marcescens LVF3 dsDNA, ssDNA, dsRNA, and ssRNA host-associated metavirome analysis. Of the 25 distinctive dsDNA phage isolates, 14 were associated with Brevundimonas and 11 with Serratia. TEM analysis revealed that 6 were myoviruses, 18 siphoviruses and 1 podovirus, while phages infecting Brevundimonas belonged all to siphoviruses. The associated viromes suggested a higher phage diversity in summer than in winter, and dsDNA phages were the dominant group. Isolation of vB_SmaP-Kaonashi was possible after investigating the viromes associated with Serratia, demonstrating the great potential of accompanying host-associated metavirome analysis. The ssDNA virome analysis showed that the B. pondensis LVF1 host is associated with Microviridae and Inoviridae phages, although none of them were isolated. The results demonstrated that the classical isolation technique is not exhausted, leading to the isolation of new dsDNA phages. It can be further improved by combination with metavirome techniques, which revealed further diversity.

Outer membrane targeting, ultrastructure, and single molecule localization of the enteropathogenic Escherichia coli type IV pilus secretin BfpB.

Type IV pili (T4P) are filamentous surface appendages required for tissue adherence, motility, aggregation, and transformation in a wide array of bacteria and archaea. The bundle-forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC) is a prototypical T4P and confirmed virulence factor. T4P fibers are assembled by a complex biogenesis machine that extrudes pili through an outer membrane (OM) pore formed by the secretin protein. Secretins constitute a superfamily of proteins that assemble into multimers and support the transport of macromolecules by four evolutionarily ancient secretion systems: T4P, type II secretion, type III secretion, and phage assembly. Here, we determine that the lipoprotein transport pathway is not required for targeting the BfpB secretin protein of the EPEC T4P to the OM and describe the ultrastructure of the single particle averaged structures of the assembled complex by transmission electron microscopy. Furthermore, we use photoactivated localization microscopy to determine the distribution of single BfpB molecules fused to photoactivated mCherry. In contrast to findings in other T4P systems, we found that BFP components predominantly have an uneven distribution through the cell envelope and are only found at one or both poles in a minority of cells. In addition, we report that concurrent mutation of both the T4bP secretin and the retraction ATPase can result in viable cells and found that these cells display paradoxically low levels of cell envelope stress response activity. These results imply that secretins can direct their own targeting, have complex distributions and provide feedback information on the state of pilus biogenesis.

Combined activity of LACS1 and LACS4 is required for proper pollen coat formation in Arabidopsis.

Very long chain lipids are important components of the plant cuticle that establishes the boundary surface of aerial organs. In addition, these lipids were detected in the extracellular pollen coat (tryphine), where they play a crucial role in appropriate pollen-stigma communication. As such they are involved in the early interaction of pollen with the stigma. A substantial reduction in tryphine lipids was shown to compromise pollen germination and, consequently, resulted in male sterility. We investigated the role of two long-chain acyl-CoA synthetases (LACSs) in Arabidopsis with respect to their contribution to the production of tryphine lipids. LACS was shown to provide CoA-activated very long chain fatty acids (VLCFA-CoAs) to the pathways of wax biosynthesis. The allocation of sufficient quantities of VLCFA-CoA precursors should therefore be relevant to the generation of tryphine lipids. Here, we report on the identification of lacs1 lacs4 double knock-out mutant lines that were conditionally sterile and showed significant reductions in pollen coat lipids. Whereas the contributions of both LACS proteins to surface wax levels were roughly additive, their co-operation in tryphine lipid biosynthesis was clearly more complex. The inactivation of LACS4 resulted in increased levels of tryphine lipids accompanied by morphological anomalies of the pollen grains. The additional inactivation of LACS1 neutralized the morphological defects, decreased the tryphine lipids far below wild-type levels and resulted in conditionally sterile pollen.

Archaea in symbioses.

During the last few years, the analysis of microbial diversity in various habitats greatly increased our knowledge on the kingdom Archaea. At the same time, we became aware of the multiple ways in which Archaea may interact with each other and with organisms of other kingdoms. The large group of euryarchaeal methanogens and their methane oxidizing relatives, in particular, take part in essential steps of the global methane cycle. Both of these processes, which are in reverse to each other, are partially conducted in a symbiotic interaction with different partners, either ciliates and xylophagous animals or sulfate reducing bacteria. Other symbiotic interactions are mostly of unknown ecological significance but depend on highly specific mechanisms. This paper will give an overview on interactions between Archaea and other organisms and will point out the ecological relevance of these symbiotic processes, as long as these have been already recognized.

Characterization of glyphosate-resistant Burkholderia anthina and Burkholderia cenocepacia isolates from a commercial Roundup® solution.

Roundup® is the brand name for herbicide solutions containing glyphosate, which specifically inhibits the 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase of the shikimate pathway. The inhibition of the EPSP synthase causes plant death because EPSP is required for biosynthesis of aromatic amino acids. Glyphosate also inhibits the growth of archaea, bacteria, Apicomplexa, algae and fungi possessing an EPSP synthase. Here, we have characterized two glyphosate-resistant bacteria from a Roundup solution. Taxonomic classification revealed that the isolates 1CH1 and 2CH1 are Burkholderia anthina and Burkholderia cenocepacia strains respectively. Both isolates cannot utilize glyphosate as a source of phosphorus and synthesize glyphosate-sensitive EPSP synthase variants. Burkholderia. anthina 1CH1 and B. cenocepacia 2CH1 tolerate high levels of glyphosate because the herbicide is not taken up by the bacteria. Previously, it has been observed that the exposure of soil bacteria to herbicides like glyphosate promotes the development of antibiotic resistances. Antibiotic sensitivity testing revealed that the only the B. cenocepacia 2CH1 isolate showed increased resistance to a variety of antibiotics. Thus, the adaptation of B. anthina 1CH1 and B. cenocepacia 2CH1 to glyphosate did not generally increase the antibiotic resistance of both bacteria. However, our study confirms the genomic adaptability of bacteria belonging to the genus Burkholderia.

The COP9 signalosome mediates transcriptional and metabolic response to hormones, oxidative stress protection and cell wall rearrangement during fungal development.

The COP9 signalosome complex (CSN) is a crucial regulator of ubiquitin ligases. Defects in CSN result in embryonic impairment and death in higher eukaryotes, whereas the filamentous fungus Aspergillus nidulans survives without CSN, but is unable to complete sexual development. We investigated overall impact of CSN activity on A. nidulans cells by combined transcriptome, proteome and metabolome analysis. Absence of csn5/csnE affects transcription of at least 15% of genes during development, including numerous oxidoreductases. csnE deletion leads to changes in the fungal proteome indicating impaired redox regulation and hypersensitivity to oxidative stress. CSN promotes the formation of asexual spores by regulating developmental hormones produced by PpoA and PpoC dioxygenases. We identify more than 100 metabolites, including orsellinic acid derivatives, accumulating preferentially in the csnE mutant. We also show that CSN is required to activate glucanases and other cell wall recycling enzymes during development. These findings suggest a dual role for CSN during development: it is required early for protection against oxidative stress and hormone regulation and is later essential for control of the secondary metabolism and cell wall rearrangement.

Metagenome-Assembled Genome Sequences of a Biofilm Derived from Marsberg Copper Mine.

We sequenced the metagenome of a biofilm collected near a leachate stream of the Marsberg copper mine (Germany) and reconstructed eight metagenome-assembled genomes. These genomes yield copper resistance through Cu(I) oxidation via multiple copper oxidases and extrusion through copper-exporting P-type ATPases.

16S rRNA Amplicon Sequencing of Microbial Biofilms from Marsberg Copper Mine, Germany.

The 16S rRNA amplicons from biofilms inhabiting rocks near various water bodies of Marsberg Copper Mine (Rhenish Massif, Germany) reveal the diversity of their microbial communities. The abundance of Chloroflexi and Cyanobacteria taxa in the biofilms near leachate streams indicated the selective enrichment of Ktedonobacteria and Oxyphotobacteria members.