Binding of GTP to BifA is required for the production of Pel-dependent biofilms in Pseudomonas aeruginosa.

The Pel exopolysaccharide is one of the most mechanistically conserved and phylogenetically diverse bacterial biofilm matrix determinants. Pel is a major contributor to the structural integrity of Pseudomonas aeruginosa biofilms, and its biosynthesis is regulated by the binding of cyclic-3',5'-dimeric guanosine monophosphate (c-di-GMP) to the PelD receptor. c-di-GMP is synthesized from two molecules of guanosine triphosphate (GTP) by diguanylate cyclases with GGDEF domains and degraded by phosphodiesterases with EAL or HD-GYP domains. As the P. aeruginosa genome encodes 43 c-di-GMP metabolic enzymes, one way signaling specificity can be achieved is through direct interaction between specific enzyme-receptor pairs. Here, we show that the inner membrane hybrid GGDEF-EAL enzyme, BifA, directly interacts with PelD via its cytoplasmic HAMP, GGDEF, and EAL domains. Despite having no catalytic function, the degenerate active site motif of the BifA GGDEF domain (GGDQF) has retained the ability to bind GTP with micromolar affinity. Mutations that abolish GTP binding result in increased biofilm formation but stable global c-di-GMP levels. Our data suggest that BifA forms a dimer in solution and that GTP binding induces conformational changes in dimeric BifA that enhance the BifA-PelD interaction and stimulate its phosphodiesterase activity, thus reducing c-di-GMP levels and downregulating Pel biosynthesis. Structural comparisons between the dimeric AlphaFold2 model of BifA and the structures of other hybrid GGDEF-EAL proteins suggest that the regulation of BifA by GTP may occur through a novel mechanism.IMPORTANCEc-di-GMP is the most common cyclic dinucleotide used by bacteria to regulate phenotypes such as motility, biofilm formation, virulence factor production, cell cycle progression, and cell differentiation. While the identification and initial characterization of c-di-GMP metabolic enzymes are well established, our understanding of how these enzymes are regulated to provide signaling specificity remains understudied. Here we demonstrate that the inactive GGDEF domain of BifA binds GTP and regulates the adjacent phosphodiesterase EAL domain, ultimately downregulating Pel-dependent P. aeruginosa biofilm formation through an interaction with PelD. This discovery adds to the growing body of literature regarding how hybrid GGDEF-EAL enzymes are regulated and provides additional precedence for studying how direct interactions between c-di-GMP metabolic enzymes and effectors result in signaling specificity.

Holtiella tumoricola gen. nov. sp. nov., isolated from a human clinical sample.

The diversity of bacteria associated with biopsy material obtained from patients with colorectal cancer was investigated using culture techniques. A novel bacterium, strain CC70AT, was isolated by diluting a sample of homogenized tissue in anaerobic medium, and then plating to yield a pure culture. Strain CC70AT was a Gram-positive, strictly anaerobic, motile, rod-shaped bacterium. Formate, but not acetate, was a fermentative end-product from growth in peptone-yeast extract and peptone-yeast-glucose broth. The G+C content of DNA from strain CC70AT was 34.9 mol%. 16S rRNA gene sequence analysis revealed that the isolate was part of the phylum Bacillota. The closest described relatives of strain CC70AT were Cellulosilyticum lentocellum (93.3 %) and Cellulosilyticum ruminicola (93.3 and 91.9% sequence similarity across 16S rRNA gene, respectively). According to the data obtained in this work, strain CC70AT represents a novel bacterium belonging to a new genus for which the name Holtiella tumoricola gen. nov., sp. nov. is proposed. The type strain for our described novel species is CC70AT (=DSM 27931T= JCM 30568T).

The Sia System and c-di-GMP Play a Crucial Role in Controlling Cell-Association of Psl in Planktonic P. aeruginosa.

Many bacterial species use the secondary messenger, c-di-GMP, to promote the production of biofilm matrix components. In Pseudomonas aeruginosa, c-di-GMP production is stimulated upon initial surface contact and generally remains high throughout biofilm growth. Transcription of several gene clusters, including the Sia signal transduction system, are induced in response to high cellular levels of c-di-GMP. The output of this system is SiaD, a diguanylate cyclase whose activity is induced in the presence of the detergent SDS. Previous studies demonstrated that Sia-mediated cellular aggregation is a key feature of P. aeruginosa growth in the presence of SDS. Here, we show that the Sia system is important for producing low levels of c-di-GMP when P. aeruginosa is growing planktonically. In addition, we show that Sia activity is important for maintaining cell-associated Psl in planktonic populations. We also demonstrate that Sia mutant strains have reduced cell-associated Psl and a surface attachment-deficient phenotype. The Sia system also appears to posttranslationally impact cell-associated Psl levels. Collectively, our findings suggest a novel role for the Sia system and c-di-GMP in planktonic populations by regulating levels of cell-associated Psl.

Mucoid Pseudomonas aeruginosa Can Produce Calcium-Gelled Biofilms Independent of the Matrix Components Psl and CdrA.

Biofilms are aggregates of microorganisms embedded in an extracellular matrix comprised largely of exopolysaccharides (EPSs), nucleic acids, and proteins. Pseudomonas aeruginosa is an opportunistic human pathogen that is also a model organism for studying biofilms in the laboratory. Here, we define a novel program of biofilm development used by mucoid (alginate-overproducing) P. aeruginosa in the presence of elevated calcium. Calcium cations cross-link negatively charged alginate polymers, resulting in individual cells being suspended in an alginate gel. The formation of this type of structurally distinct biofilm is not reliant on the canonical biofilm EPS components Psl and Pel or the matrix protein CdrA. We also observed that mucoid P. aeruginosa biofilm cells do not have the typical elevated levels of the secondary messenger cyclic di-GMP (c-di-GMP), as expected of biofilm cells, nor does the overproduction of alginate rely on high c-di-GMP. This contrasts with nonmucoid biofilms in which the production of the matrix components Psl, Pel, and CdrA is positively regulated by elevated c-di-GMP. We further demonstrate that calcium-gelled alginate biofilms impede the penetration of the antibiotic tobramycin, thus protecting the biofilm community from antibiotic-mediated killing. Finally, we show that bacterial aggregates with a dispersed cell arrangement like laboratory-grown calcium-alginate biofilm structures are present in explanted cystic fibrosis (CF) lung samples. Our findings illustrate the diverse nature of biofilm formation and structure in P. aeruginosa. IMPORTANCE The opportunistic pathogen Pseudomonas aeruginosa produces a complex biofilm matrix comprised of exopolysaccharides (EPSs), nucleic acids, and proteins. P. aeruginosa biofilm formation canonically depends on a variable combination of the exopolysaccharides Psl and Pel and the matrix protein CdrA. We demonstrate that mucoid P. aeruginosa, which overproduces the EPS alginate, possesses an entirely alternate and calcium-dependent method of biofilm formation. These mucoid biofilm structures do not require Psl, Pel, or CdrA, and they display a unique organization of individually suspended cells similar to bacterial aggregates observed in cystic fibrosis airways. Furthermore, calcium-gelled mucoid biofilms impede the penetration and killing action of the antibiotic tobramycin, illustrating their potential clinical significance. Our findings highlight the compositional and structural variety of P. aeruginosa biofilm aggregates.

The Wsp system of Pseudomonas aeruginosa links surface sensing and cell envelope stress.

Surface sensing is a critical process that promotes the transition to a biofilm lifestyle. Several surface-sensing mechanisms have been described for a range of species, most involving surface appendages, such as flagella and pili. Pseudomonas aeruginosa uses the Wsp chemosensory-like signal transduction pathway to sense surfaces and promote biofilm formation. The methyl-accepting chemotaxis protein WspA recognizes an unknown surface-associated signal and initiates a phosphorylation cascade that activates the diguanylate cyclase WspR. We conducted a screen for Wsp-activating compounds and found that chemicals that impact the cell envelope induce Wsp signaling, increase intracellular c-di-GMP levels, and can promote surface attachment. To isolate the Wsp system from other P. aeruginosa surface-sensing systems, we heterologously expressed it in Escherichia coli and found it sufficient for sensing surfaces and the chemicals identified in our screen. Using well-characterized reporters for different E. coli cell envelope stress responses, we then determined that Wsp sensitivity overlapped with multiple E. coli cell envelope stress-response systems. Using mutational and CRISPRi analysis, we found that misfolded proteins in the periplasm appear to be a major stimulus of the Wsp system. Finally, we show that surface attachment appears to have an immediate, observable effect on cell envelope integrity. Collectively, our results provide experimental evidence that cell envelope stress represents an important feature of surface sensing in P. aeruginosa.

L-Arabinose Transport and Metabolism in Salmonella Influences Biofilm Formation.

L-arabinose inducible promoters are commonly used in gene expression analysis. However, nutrient source and availability also play a role in biofilm formation; therefore, L-arabinose metabolism could impact biofilm development. In this study we examined the impact of L-arabinose on Salmonella enterica serovar Typhimurium (S. Typhimurium) biofilm formation. Using mutants impaired for the transport and metabolism of L-arabinose, we showed that L-arabinose metabolism negatively impacts S. Typhimurium biofilm formation in vitro. When L-arabinose metabolism is abrogated, biofilm formation returned to baseline levels. However, without the ability to import extracellular L-arabinose, biofilm formation significantly increased. Using RNA-Seq we identified several gene families involved in these different phenotypes including curli expression, amino acid synthesis, and L-arabinose metabolism. Several individual candidate genes were tested for their involvement in the L-arabinose-mediated biofilm phenotypes, but most played no significant role. Interestingly, in the presence of L-arabinose the diguanylate cyclase gene adrA was downregulated in wild type S. Typhimurium. Meanwhile cyaA, encoding an adenylate cyclase, was downregulated in an L-arabinose transport mutant. Using an IPTG-inducible plasmid to deplete c-di-GMP via vieA expression, we were able to abolish the increased biofilm phenotype seen in the transport mutant. However, the mechanism by which the L-arabinose import mutant forms significantly larger biofilms remains to be determined. Regardless, these data suggest that L-arabinose metabolism influences intracellular c-di-GMP levels and therefore biofilm formation. These findings are important when considering the use of an L-arabinose inducible promoter in biofilm conditions.

Rural exclusion from science and academia.

Rural individuals are underrepresented in science at all levels. The disenfranchisement of rural people in science and research foments a cultural divide between rural America and the scientific community. Science can improve inclusion of rural individuals by removing barriers in academia that disfavor those from first-generation and low-socioeconomic backgrounds.

Prevalence and Epidemiology of Mycoplasma genitalium in a Pacific-Region Military Population.

BACKGROUND: Mycoplasma genitalium is an important emerging sexually transmitted pathogen commonly causing urethritis in men, cervicitis, and pelvic inflammatory disease in women with potential of infertility. Accumulating evidence identifies the prevalence of M. genitalium similar to long recognized pathogens, Chlamydia trachomatis and Neisseria gonorrhoeae. The purpose of this study was to establish the prevalence and epidemiology of M. genitalium in a mid-Pacific military population. METHODS: A prospective analysis was conducted from routine specimens collected as standard of care for sexually transmitted infection (STI) testing at Tripler Army Medical Center on Oahu, HI. The prevalence of M. genitalium was determined using the Aptima M. genitalium assay, a transcription-mediated amplification test. A multivariate analysis was performed to assess the associations for this infection with other STIs and demographic factors. RESULTS: A total of 1876 specimens were tested in a 6-month period including 6 sample types from 1158 females and 718 males. Subject ages ranged from 18 to 76 years, with a median of 24 years (interquartile range, 21-29 years). The prevalence of M. genitalium was 8.8% overall (n = 165), 7.1% in females and 11.6% in males. Coinfection with M. genitalium occurred with another sexually-transmitted pathogen in 43 patients (18.3%), with C. trachomatis as the most common organism (n = 38). CONCLUSIONS: These data contribute to the evidence base for M. genitalium and STI screening in an active-duty military.

Specific Root Exudate Compounds Sensed by Dedicated Chemoreceptors Shape Azospirillum brasilense Chemotaxis in the Rhizosphere.

Plant roots shape the rhizosphere community by secreting compounds that recruit diverse bacteria. Colonization of various plant roots by the motile alphaproteobacterium Azospirillum brasilense causes increased plant growth, root volume, and crop yield. Bacterial chemotaxis in this and other motile soil bacteria is critical for competitive colonization of the root surfaces. The role of chemotaxis in root surface colonization has previously been established by endpoint analyses of bacterial colonization levels detected a few hours to days after inoculation. More recently, microfluidic devices have been used to study plant-microbe interactions, but these devices are size limited. Here, we use a novel slide-in chamber that allows real-time monitoring of plant-microbe interactions using agriculturally relevant seedlings to characterize how bacterial chemotaxis mediates plant root surface colonization during the association of A. brasilense with Triticum aestivum (wheat) and Medicago sativa (alfalfa) seedlings. We track A. brasilense accumulation in the rhizosphere and on the root surfaces of wheat and alfalfa. A. brasilense motile cells display distinct chemotaxis behaviors in different regions of the roots, including attractant and repellent responses that ultimately drive surface colonization patterns. We also combine these observations with real-time analyses of behaviors of wild-type and mutant strains to link chemotaxis responses to distinct chemicals identified in root exudates to specific chemoreceptors that together explain the chemotactic response of motile cells in different regions of the roots. Furthermore, the bacterial second messenger c-di-GMP modulates these chemotaxis responses. Together, these findings illustrate dynamic bacterial chemotaxis responses to rhizosphere gradients that guide root surface colonization.IMPORTANCE Plant root exudates play critical roles in shaping rhizosphere microbial communities, and the ability of motile bacteria to respond to these gradients mediates competitive colonization of root surfaces. Root exudates are complex chemical mixtures that are spatially and temporally dynamic. Identifying the exact chemical(s) that mediates the recruitment of soil bacteria to specific regions of the roots is thus challenging. Here, we connect patterns of bacterial chemotaxis responses and sensing by chemoreceptors to chemicals found in root exudate gradients and identify key chemical signals that shape root surface colonization in different plants and regions of the roots.

Bridging the Gap Between Single-Strain and Community-Level Plant-Microbe Chemical Interactions.

Although the influence of microbiomes on the health of plant hosts is evident, specific mechanisms shaping the structure and dynamics of microbial communities in the phyllosphere and rhizosphere are only beginning to become clear. Traditionally, plant-microbe interactions have been studied using cultured microbial isolates and plant hosts but the rising use of 'omics tools provides novel snapshots of the total complex community in situ. Here, we discuss the recent advances in tools and techniques used to monitor plant-microbe interactions and the chemical signals that influence these relationships in above- and belowground tissues. Particularly, we highlight advances in integrated microscopy that allow observation of the chemical exchange between individual plant and microbial cells, as well as high-throughput, culture-independent approaches to investigate the total genetic and metabolic contribution of the community. The chemicals discussed have been identified as relevant signals across experimental spectrums. However, mechanistic insight into the specific interactions mediated by many of these chemicals requires further testing. Experimental designs that attempt to bridge the gap in biotic complexity between single strains and whole communities will advance our understanding of the chemical signals governing plant-microbe associations in the rhizosphere and phyllosphere.

Distinct Chemotaxis Protein Paralogs Assemble into Chemoreceptor Signaling Arrays To Coordinate Signaling Output.

Most chemotactic motile bacteria possess multiple chemotaxis signaling systems, the functions of which are not well characterized. Chemotaxis signaling is initiated by chemoreceptors that assemble as large arrays, together with chemotaxis coupling proteins (CheW) and histidine kinase proteins (CheA), which form a baseplate with the cytoplasmic tips of receptors. These cell pole-localized arrays mediate sensing, signaling, and signal amplification during chemotaxis responses. Membrane-bound chemoreceptors with different cytoplasmic domain lengths segregate into distinct arrays. Here, we show that a bacterium, Azospirillum brasilense, which utilizes two chemotaxis signaling systems controlling distinct motility parameters, coordinates its chemotactic responses through the production of two separate membrane-bound chemoreceptor arrays by mixing paralogs within chemotaxis baseplates. The polar localization of chemoreceptors of different length classes is maintained in strains that had baseplate signaling proteins from either chemotaxis system but was lost when both systems were deleted. Chemotaxis proteins (CheA and CheW) from each of the chemotaxis signaling systems (Che1 and Che4) could physically interact with one another, and chemoreceptors from both classes present in A. brasilense could interact with Che1 and Che4 proteins. The assembly of paralogs from distinct chemotaxis pathways into baseplates provides a straightforward mechanism for coordinating signaling from distinct pathways, which we predict is not unique to this system given the propensity of chemotaxis systems for horizontal gene transfer.IMPORTANCE The assembly of chemotaxis receptors and signaling proteins into polar arrays is universal in motile chemotactic bacteria. Comparative genome analyses indicate that most motile bacteria possess multiple chemotaxis signaling systems, and experimental evidence suggests that signaling from distinct chemotaxis systems is integrated. Here, we identify one such mechanism. We show that paralogs from two chemotaxis systems assemble together into chemoreceptor arrays, forming baseplates comprised of proteins from both chemotaxis systems. These mixed arrays provide a straightforward mechanism for signal integration and coordinated response output from distinct chemotaxis systems. Given that most chemotactic bacteria encode multiple chemotaxis systems and the propensity for these systems to be laterally transferred, this mechanism may be common to ensure chemotaxis signal integration occurs.

Modeling aerotaxis band formation in Azospirillum brasilense.

BACKGROUND: Bacterial chemotaxis, the ability of motile bacteria to navigate gradients of chemicals, plays key roles in the establishment of various plant-microbe associations, including those that benefit plant growth and crop productivity. The motile soil bacterium Azospirillum brasilense colonizes the rhizosphere and promotes the growth of diverse plants across a range of environments. Aerotaxis, or the ability to navigate oxygen gradients, is a widespread behavior in bacteria. It is one of the strongest behavioral responses in A. brasilense and it is essential for successful colonization of the root surface. Oxygen is one of the limiting nutrients in the rhizosphere where density and activity of organisms are greatest. The aerotaxis response of A. brasilense is also characterized by high precision with motile cells able to detect narrow regions in a gradient where the oxygen concentration is low enough to support their microaerobic lifestyle and metabolism. RESULTS: Here, we present a mathematical model for aerotaxis band formation that captures most critical features of aerotaxis in A. brasilense. Remarkably, this model recapitulates experimental observations of the formation of a stable aerotactic band within 2 minutes of exposure to the air gradient that were not captured in previous modeling efforts. Using experimentally determined parameters, the mathematical model reproduced an aerotactic band at a distance from the meniscus and with a width that matched the experimental observation. CONCLUSIONS: Including experimentally determined parameter values allowed us to validate a mathematical model for aerotactic band formation in spatial gradients that recapitulates the spatiotemporal stability of the band and its position in the gradient as well as its overall width. This validated model also allowed us to capture the range of oxygen concentrations the bacteria prefer during aerotaxis, and to estimate the effect of parameter values (e.g. oxygen consumption rate), both of which are difficult to obtain in experiments.

A PilZ-Containing Chemotaxis Receptor Mediates Oxygen and Wheat Root Sensing in Azospirillum brasilense.

Chemotactic bacteria sense environmental changes via dedicated receptors that bind to extra- or intracellular cues and relay this signal to ultimately alter direction of movement toward beneficial cues and away from harmful environments. In complex environments, such as the rhizosphere, bacteria must be able to sense and integrate diverse cues. Azospirillum brasilense is a microaerophilic motile bacterium that promotes growth of cereals and grains. Root surface colonization is a prerequisite for the beneficial effects on plant growth but how motile A. brasilense navigates the rhizosphere is poorly studied. Previously only 2 out of 51 A. brasilense chemotaxis receptors have been characterized, AerC and Tlp1, and only Tlp1 was found to be essential for wheat root colonization. Here we describe another chemotaxis receptor, named Aer, that is homologous to the Escherichia coli Aer receptor, likely possesses an FAD cofactor and is involved in aerotaxis (taxis in an air gradient). We also found that the A. brasilense Aer contributes to sensing chemical gradients originating from wheat roots. In addition to A. brasilense Aer having a putative N-terminal FAD-binding PAS domain, it possesses a C-terminal PilZ domain that contains all the conserved residues for binding c-di-GMP. Mutants lacking the PilZ domain of Aer are altered in aerotaxis and are completely null in wheat root colonization and they also fail to sense gradients originating from wheat roots. The PilZ domain of Aer is also vital in integrating Aer signaling with signaling from other chemotaxis receptors to sense gradients from wheat root surfaces and colonizing wheat root surfaces.

Analyzing Chemotaxis and Related Behaviors of Azospirillum Brasilense.

Bacteria of the genus A. brasilense are motile and capable of chemotaxis and aerotaxis (taxis in gradient of oxygen) using a single polar flagellum that propels the cells in aqueous environments. Responses to attractants and repellents have been described and spatial gradient assays that permit the visualization of these responses are detailed in this unit. These assays are simple and can be readily implemented with minimum set ups. © 2018 by John Wiley & Sons, Inc.

Azospirillum brasilense: Laboratory Maintenance and Genetic Manipulation.

Bacteria of the genus Azospirillum, including the most comprehensively studied Azospirillum brasilense, are non-pathogenic soil bacteria that promote the growth of diverse plants, making them an attractive model to understand non-symbiotic, beneficial plant-bacteria associations. Research into the physiology and genetics of these organisms spans decades and a range of molecular tools and protocols have been developed for allelic exchange mutagenesis, in trans expression of genes, and fusions to reporter genes. © 2017 by John Wiley & Sons, Inc.

Using Light-Activated Enzymes for Modulating Intracellular c-di-GMP Levels in Bacteria.

Signaling pathways involving second messenger c-di-GMP regulate various aspects of bacterial physiology and behavior. We describe the use of a red light-activated diguanylate cyclase (c-di-GMP synthase) and a blue light-activated c-di-GMP phosphodiesterase (hydrolase) for manipulating intracellular c-di-GMP levels in bacterial cells. We illustrate the application of these enzymes in regulating several c-di-GMP-dependent phenotypes, i.e., motility and biofilm phenotypes in E. coli and chemotactic behavior in the alphaproteobacterium Azospirillum brasilense. We expect these light-activated enzymes to be also useful in regulating c-di-GMP-dependent processes occurring at the fast timescale, for spatial control of bacterial populations, as well as for analyzing c-di-GMP-dependent phenomena at the single-cell level.

Optogenetic Manipulation of Cyclic Di-GMP (c-di-GMP) Levels Reveals the Role of c-di-GMP in Regulating Aerotaxis Receptor Activity in Azospirillum brasilense.

Bacterial chemotaxis receptors provide the sensory inputs that inform the direction of navigation in changing environments. Recently, we described the bacterial second messenger cyclic di-GMP (c-di-GMP) as a novel regulator of a subclass of chemotaxis receptors. In Azospirillum brasilense, c-di-GMP binds to a chemotaxis receptor, Tlp1, and modulates its signaling function during aerotaxis. Here, we further characterize the role of c-di-GMP in aerotaxis using a novel dichromatic optogenetic system engineered for manipulating intracellular c-di-GMP levels in real time. This system comprises a red/near-infrared-light-regulated diguanylate cyclase and a blue-light-regulated c-di-GMP phosphodiesterase. It allows the generation of transient changes in intracellular c-di-GMP concentrations within seconds of irradiation with appropriate light, which is compatible with the time scale of chemotaxis signaling. We provide experimental evidence that binding of c-di-GMP to the Tlp1 receptor activates its signaling function during aerotaxis, which supports the role of transient changes in c-di-GMP levels as a means of adjusting the response of A. brasilense to oxygen gradients. We also show that intracellular c-di-GMP levels in A. brasilense change with carbon metabolism. Our data support a model whereby c-di-GMP functions to imprint chemotaxis receptors with a record of recent metabolic experience, to adjust their contribution to the signaling output, thus allowing the cells to continually fine-tune chemotaxis sensory perception to their metabolic state.IMPORTANCE Motile bacteria use chemotaxis to change swimming direction in response to changes in environmental conditions. Chemotaxis receptors sense environmental signals and relay sensory information to the chemotaxis machinery, which ultimately controls the swimming pattern of cells. In bacteria studied to date, differential methylation has been known as a mechanism to control the activity of chemotaxis receptors and modulates their contribution to the overall chemotaxis response. Here, we used an optogenetic system to perturb intracellular concentrations of the bacterial second messenger c-di-GMP to show that in some chemotaxis receptors, c-di-GMP functions in a similar feedback loop to connect the metabolic status of the cells to the sensory activity of chemotaxis receptors.

Luethyella okanaganae gen. nov., sp. nov., a Novel Genus and Species of the Family Microbacteriaceae Isolated from the Insect Okanagana rimosa.

The entomopathogen "Corynebacterium okanaganae" was described by Lüthy in 1974 but the name was never validly published. Phylogenetic analysis employing 16S rRNA gene sequences demonstrate that "Corynebacterium okanaganae" is not a member of the genus Corynebacterium but related to members of the Microbacteriaceae being most closely related to, but distinct from, members of the genera Rathayibacter, Mycetocola and Curtobacterium. The bacterium is an aerobic, Gram-positive staining, rod-shaped actinobacterium with the cell-wall peptidoglycan based on 2,4, diaminobutyric acid as the diagnostic diamino acid. The predominant menaquinones are MK-10, MK-11 and MK-12, and the principle polar lipids are phosphatidylglycerol and diphosphatidylglycerol. The major fatty acids consist of anteiso-C15:0 and anteiso-C17:0. Therefore, based upon the phylogenetic, biochemical, and chemotaxonomic information, the organism merits recognition as a novel species and genus in the family Microbacteriaceae, for which the name Luethyella okanaganae gen. nov. sp. nov. is proposed. The type strain is LBG B4405T = CCUG 43304T = NCIMB 702272T.

Peptoniphilus catoniae sp. nov., isolated from a human faecal sample from a traditional Peruvian coastal community.

A novel Gram-stain-positive, coccus-shaped, obligately anaerobic bacterium was isolated from a faecal sample obtained from an individual in a traditional community located off the southern coast of Peru. Comparative 16S rRNA gene sequence analysis showed the novel bacterium belonged to the genus Peptoniphilus but showed no particular relationship with any species, demonstrating less than 91 % 16S rRNA gene sequence similarity with all members of the genus. The major cellular fatty acids of the novel isolate were determined to be C10 : 0, C14 : 0, C16 : 0, C18 : 1ω9c and C18 : 2ω6,9c/anteiso-C18 : 0. The DNA G+C content was 34.4 mol%. End-products of metabolism from peptone-yeast-glucose broth (PYG) were determined to be acetate and butyrate. Based on the phenotypic, chemotaxonomic and phylogenetic results, the organism represents a novel species of the genus Peptoniphilus, for which the name Peptoniphilus catoniae sp. nov. is proposed. The type strain is M6.X2DT ( = DSM 29874T = CCUG 66798T).

Clostridium amazonense sp. nov. an obliqately anaerobic bacterium isolated from a remote Amazonian community in Peru.

A strictly anaerobic Gram-stain positive, spore-forming, rod-shaped bacterium designated NE08V(T), was isolated from a fecal sample of an individual residing in a remote Amazonian community in Peru. Phylogenetic analysis based on the 16S rRNA gene sequence showed the organism belonged to the genus Clostridium and is most closely related to Clostridium vulturis (97.4% sequence similarity) and was further characterized using biochemical and chemotaxonomic methods. The major cellular fatty acids were anteiso C13:0 and C16:0 with a genomic DNA G + C content of 31.6 mol%. Fermentation products during growth with PYG were acetate and butyrate. Based on phylogenetic, phenotypic and chemotaxonomic information, strain NE08V was identified as representing a novel species of the genus Clostridium, for which the name Clostridium amazonense sp. nov. is proposed. The type strain is NE08V(T) (DSM 23598(T) = CCUG 59712(T)).