Intragenomic conflicts with plasmids and chromosomal mobile genetic elements drive the evolution of natural transformation within species.

Natural transformation is the only mechanism of genetic exchange controlled by the recipient bacteria. We quantified its rates in 786 clinical strains of the human pathogens Legionella pneumophila (Lp) and 496 clinical and environmental strains of Acinetobacter baumannii (Ab). The analysis of transformation rates in the light of phylogeny revealed they evolve by a mixture of frequent small changes and a few large quick jumps across 6 orders of magnitude. In standard conditions close to half of the strains of Lp and a more than a third in Ab are below the detection limit and thus presumably non-transformable. Ab environmental strains tend to have higher transformation rates than the clinical ones. Transitions to non-transformability were frequent and usually recent, suggesting that they are deleterious and subsequently purged by natural selection. Accordingly, we find that transformation decreases genetic linkage in both species, which might accelerate adaptation. Intragenomic conflicts with chromosomal mobile genetic elements (MGEs) and plasmids could explain these transitions and a GWAS confirmed systematic negative associations between transformation and MGEs: plasmids and other conjugative elements in Lp, prophages in Ab, and transposable elements in both. In accordance with the hypothesis of modulation of transformation rates by genetic conflicts, transformable strains have fewer MGEs in both species and some MGEs inactivate genes implicated in the transformation with heterologous DNA (in Ab). Innate defense systems against MGEs are associated with lower transformation rates, especially restriction-modification systems. In contrast, CRISPR-Cas systems are associated with higher transformation rates suggesting that adaptive defense systems may facilitate cell protection from MGEs while preserving genetic exchanges by natural transformation. Ab and Lp have different lifestyles, gene repertoires, and population structure. Nevertheless, they exhibit similar trends in terms of variation of transformation rates and its determinants, suggesting that genetic conflicts could drive the evolution of natural transformation in many bacteria.

Evolutionarily stable gene clusters shed light on the common grounds of pathogenicity in the Acinetobacter calcoaceticus-baumannii complex.

Nosocomial pathogens of the Acinetobacter calcoaceticus-baumannii (ACB) complex are a cautionary example for the world-wide spread of multi- and pan-drug resistant bacteria. Aiding the urgent demand for novel therapeutic targets, comparative genomics studies between pathogens and their apathogenic relatives shed light on the genetic basis of human-pathogen interaction. Yet, existing studies are limited in taxonomic scope, sensing of the phylogenetic signal, and resolution by largely analyzing genes independent of their organization in functional gene clusters. Here, we explored more than 3,000 Acinetobacter genomes in a phylogenomic framework integrating orthology-based phylogenetic profiling and microsynteny conservation analyses. We delineate gene clusters in the type strain A. baumannii ATCC 19606 whose evolutionary conservation indicates a functional integration of the subsumed genes. These evolutionarily stable gene clusters (ESGCs) reveal metabolic pathways, transcriptional regulators residing next to their targets but also tie together sub-clusters with distinct functions to form higher-order functional modules. We shortlisted 150 ESGCs that either co-emerged with the pathogenic ACB clade or are preferentially found therein. They provide a high-resolution picture of genetic and functional changes that coincide with the manifestation of the pathogenic phenotype in the ACB clade. Key innovations are the remodeling of the regulatory-effector cascade connecting LuxR/LuxI quorum sensing via an intermediate messenger to biofilm formation, the extension of micronutrient scavenging systems, and the increase of metabolic flexibility by exploiting carbon sources that are provided by the human host. We could show experimentally that only members of the ACB clade use kynurenine as a sole carbon and energy source, a substance produced by humans to fine-tune the antimicrobial innate immune response. In summary, this study provides a rich and unbiased set of novel testable hypotheses on how pathogenic Acinetobacter interact with and ultimately infect their human host. It is a comprehensive resource for future research into novel therapeutic strategies.

Genomic plasticity and adaptive capacity of the quaternary alkyl-ammonium compound and copper tolerant Acinetobacter bohemicus strain QAC-21b isolated from pig manure.

Here, we present the genomic characterization of an Acinetobacter bohemicus strain QAC-21b which was isolated in the presence of a quaternary alky-ammonium compound (QAAC) from manure of a conventional German pig farm. The genetic determinants for QAAC, heavy metal and antibiotic resistances are reported based of the whole genome shotgun sequence and physiological growth tests. A. bohemicus QAC-21b grew in a species typical manner well at environmental temperatures but not at 37 °C. The strain showed tolerance to QAACs and copper but was susceptible to antibiotics relevant for Acinetobacter treatments. The genome of QAC-21b contained several Acinetobacter typical QAAC and heavy metal transporting efflux pumps coding genes, but no key genes for acquired antimicrobial resistances. The high genomic content of transferable genetic elements indicates that this bacterium can be involved in the transmission of antimicrobial resistances, if it is released with manure as organic fertilizer on agricultural fields. The genetic content of the strain was compared to that of two other A. bohemicus strains, the type strain ANC 3994T, isolated from forest soil, and KCTC 42081, originally described as A. pakistanensis, a metal resistant strain isolated from a wastewater treatment pond. In contrast to the forest soil strain, both strains from anthropogenically impacted sources showed genetic features indicating their evolutionary adaptation to the anthropogenically impacted environments. Strain QAC-21b will be used as model strain to study the transmission of antimicrobial resistance to environmentally adapted Acinetobacter in agricultural environments receiving high content of pollutants with organic fertilizers from livestock husbandry.

An Unprecedented Tolerance to Deletion of the Periplasmic Chaperones SurA, Skp, and DegP in the Nosocomial Pathogen Acinetobacter baumannii.

The outer membrane (OM) of Gram-negative bacteria efficiently protects from harmful environmental stresses such as antibiotics, disinfectants, or dryness. The main constituents of the OM are integral OM ß-barrel proteins (OMPs). In Gram-negative bacteria such as Escherichia coli, Yersinia enterocolitica, and Pseudomonas aeruginosa, the insertion of OMPs depends on a sophisticated biogenesis pathway. This comprises the SecYEG translocon, which enables inner membrane (IM) passage; the chaperones SurA, Skp, and DegP, which facilitate the passage of ß-barrel OMPs through the periplasm; and the ß-barrel assembly machinery (BAM), which facilitates insertion into the OM. In E. coli, Y. enterocolitica, and P. aeruginosa, the deletion of SurA is particularly detrimental and leads to a loss of OM integrity, sensitization to antibiotic treatment, and reduced virulence. In search of targets that could be exploited to develop compounds that interfere with OM integrity in Acinetobacter baumannii, we employed the multidrug-resistant strain AB5075 to generate single gene knockout strains lacking individual periplasmic chaperones. In contrast to E. coli, Y. enterocolitica, and P. aeruginosa, AB5075 tolerates the lack of SurA, Skp, or DegP with only weak mutant phenotypes. While the double knockout strains ΔsurAΔskp and ΔsurAΔdegP are conditionally lethal in E. coli, all double deletions were well tolerated by AB5075. Strikingly, even a triple-knockout strain of AB5075, lacking surA, skp, and degP, was viable. IMPORTANCE Acinetobacter baumannii is a major threat to human health due to its ability to persist in the hospital environment, resistance to antibiotic treatment, and ability to deploy multiple and redundant virulence factors. In a rising number of cases, infections with multidrug-resistant A. baumannii end up fatally, because all antibiotic treatment options fail. Thus, novel targets have to be identified and alternative therapeutics have to be developed. The knockout of periplasmic chaperones has previously proven to significantly reduce virulence and even break antibiotic resistance in other Gram-negative pathogens. Our study in A. baumannii demonstrates how variable the importance of the periplasmic chaperones SurA, Skp, and DegP can be and suggests the existence of mechanisms allowing A. baumannii to cope with the lack of the three periplasmic chaperones.

Recombinant AcnB, NrdR and RibD of Acinetobacter baumannii and their potential interaction with DNA adenine methyltransferase AamA.

In the last decades Acinetobacter baumannii developed into an increasingly challenging nosocomial pathogen. A. baumannii ATCC 17978 harbors a DNA-(adenine N6)-methyltransferase termed AamA. Previous studies revealed a low specific activity of AamA in vitro despite proven folding, which led us to speculate about possible interaction partners assisting AamA in targeting methylation sites. Here, applying a pulldown assay with subsequent mass spectrometry we identified aconitate hydratase 2 (AcnB) as possible interaction partner. In addition, we considered the putative transcriptional regulator gene nrdR (A1S_0220) and the pyrimidine deaminase/reductase gene ribD (A1S_0221) of A. baumannii strain ATCC 17978 to encode additional potential interaction partners due to their vicinity to the aamA gene (A1S_0222). Proteins were recombinantly produced in the milligram scale, purified to near homogeneity, and interactions with AamA were studied applying blue native gel electrophoreses, electrophoretic mobility shift assay, chemical cross-linking and co-immunoprecipitation. These analyses did not provide evidence of interaction between AamA and purified proteins. Solution structures of RibD, NrdR and AcnB were studied by small-angle X-ray scattering (SAXS) alone and in combination with AamA. While in the case of RibD and AcnB no evidence of an interaction with AamA was produced, addition of AamA to NrdR resulted in dissociation of long and rod-shaped polymeric NrdR structures, implying a specific but transient interaction. Moreover, we identified a molecular crowding effect possibly impeding the DNA methyltransferase activity in vivo and a sequence-independent DNA binding activity of AamA calling for continued efforts to identify the interaction network of AamA.

Acinetobacter stercoris sp. nov. isolated from output source of a mesophilic german biogas plant with anaerobic operating conditions.

The Gram-stain-negative, oxidase negative, catalase positive strain KPC-SM-21T, isolated from a digestate of a storage tank of a mesophilic German biogas plant, was investigated by a polyphasic taxonomic approach. Phylogenetic identification based on the nearly full-length 16S rRNA gene revealed highest gene sequence similarity to Acinetobacter baumannii ATCC 19606T (97.0%). Phylogenetic trees calculated based on partial rpoB and gyrB gene sequences showed a distinct clustering of strain KPC-SM-21T with Acinetobacter gerneri DSM 14967T = CIP 107464T and not with A. baumannii, which was also supported in the five housekeeping genes multilocus sequence analysis based phylogeny. Average nucleotide identity values between whole genome sequences of strain KPC-SM-21T and next related type strains supported the novel species status. The DNA G + C content of strain KPC-SM-21T was 37.7 mol%. Whole-cell MALDI-TOF MS analysis supported the distinctness of the strain to type strains of next related Acinetobacter species. Predominant fatty acids were C18:1 ω9c (44.2%), C16:0 (21.7%) and a summed feature comprising C16:1 ω7c and/or iso-C15:0 2-OH (15.3%). Based on the obtained genotypic, phenotypic and chemotaxonomic data we concluded that strain KPC-SM-21T represents a novel species of the genus Acinetobacter, for which the name Acinetobacter stercoris sp. nov. is proposed. The type strain is KPC-SM-21T (= DSM 102168T = LMG 29413T).

Novel Genes Required for Surface-Associated Motility in Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic and increasingly multi-drug resistant human pathogen rated as a critical priority one pathogen for the development of new antibiotics by the WHO in 2017. Despite the lack of flagella, A. baumannii can move along wet surfaces in two different ways: via twitching motility and surface-associated motility. While twitching motility is known to depend on type IV pili, the mechanism of surface-associated motility is poorly understood. In this study, we established a library of 30 A. baumannii ATCC® 17978™ mutants that displayed deficiency in surface-associated motility. By making use of natural competence, we also introduced these mutations into strain 29D2 to differentiate strain-specific versus species-specific effects of mutations. Mutated genes were associated with purine/pyrimidine/folate biosynthesis (e.g. purH, purF, purM, purE), alarmone/stress metabolism (e.g. Ap4A hydrolase), RNA modification/regulation (e.g. methionyl-tRNA synthetase), outer membrane proteins (e.g. ompA), and genes involved in natural competence (comEC). All tested mutants originally identified as motility-deficient in strain ATCC® 17978™ also displayed a motility-deficient phenotype in 29D2. By contrast, further comparative characterization of the mutant sets of both strains regarding pellicle biofilm formation, antibiotic resistance, and virulence in the Galleria mellonella infection model revealed numerous strain-specific mutant phenotypes. Our studies highlight the need for comparative analyses to characterize gene functions in A. baumannii and for further studies on the mechanisms underlying surface-associated motility.

The status of the genus Prolinoborus (Pot et al. 1992) and the species Prolinoborus fasciculus (Pot et al. 1992).

On the basis of two other publications (Yarza et al. 2013; Nemec et al. 2019) and on the basis of resequencing of the 16S rRNA gene of Prolinoborus fasciculus CIP 103579T it is concluded that Prolinoborus fasciculus CIP 103579T, which is the only available strain of the species from culture collections, does not conform to the original description given by Pot et al. (1992). The strain investigated is a member of the genus Acinetobacter within the Moraxellaceae, a family of the Gammaproteobacteria and not a member of the Betaproteobacteria as originally proposed. Prolinoborus fasciculus CIP 103579T shared 99.8 % 16S rRNA gene sequence similarity with Acinetobacter lwoffii DSM 2403T. The two strains clustered together by rpoB- and core genome-based phylogenetic analyses and shared an average nucleotide identity of 96.47% (reciprocal, 96.56 %) and a digital genome distance calculation (GGDC) value of 66.9 %. Furthermore, the two strains shared matrix-assisted laser desorption/ionization time of flight MS profiles to a high extent and showed highly similar cellular fatty acid profiles and physiological substrate utilization patterns. It is proposed that the Judicial commission consider (1) that the strain currently deposited as CIP 103579 be recognized as a member of Acinetobacter lwoffii; (2) placing Prolinoborus fasciculus (Pot et al. 1992) on the list of rejected names if a suitable replacement strain, or a neotype strain cannot be found within 2 years of publication of this request; and (3) place the genus name Prolinoborus (Pot et al. 1992) on the list of rejected names [Recommendation 20D (3) of the Code].

Fluorescence-Based Detection of Natural Transformation in Drug-Resistant Acinetobacter baumannii.

Acinetobacter baumannii is a nosocomial agent with a high propensity for developing resistance to antibiotics. This ability relies on horizontal gene transfer mechanisms occurring in the Acinetobacter genus, including natural transformation. To study natural transformation in bacteria, the most prevalent method uses selection for the acquisition of an antibiotic resistance marker in a target chromosomal locus by the recipient cell. Most clinical isolates of A. baumannii are resistant to multiple antibiotics, limiting the use of such selection-based methods. Here, we report the development of a phenotypic and selection-free method based on flow cytometry to detect transformation events in multidrug-resistant (MDR) clinical A. baumannii isolates. To this end, we engineered a translational fusion between the abundant and conserved A. baumannii nucleoprotein (HU) and the superfolder green fluorescent protein (sfGFP). The new method was benchmarked against the conventional antibiotic selection-based method. Using this new method, we investigated several parameters affecting transformation efficiencies and identified conditions of transformability one hundred times higher than those previously reported. Using optimized transformation conditions, we probed natural transformation in a set of MDR clinical and nonclinical animal A. baumannii isolates. Regardless of their origin, the majority of the isolates displayed natural transformability, indicative of a conserved trait in the species. Overall, this new method and optimized protocol will greatly facilitate the study of natural transformation in the opportunistic pathogen A. baumanniiIMPORTANCE Antibiotic resistance is a pressing global health concern with the rise of multiple and panresistant pathogens. The rapid and unfailing resistance to multiple antibiotics of the nosocomial agent Acinetobacter baumannii, notably to carbapenems, prompt to understand the mechanisms behind acquisition of new antibiotic resistance genes. Natural transformation, one of the horizontal gene transfer mechanisms in bacteria, was only recently described in A. baumannii and could explain its ability to acquire resistance genes. We developed a reliable method to probe and study natural transformation mechanism in A. baumannii More broadly, this new method based on flow cytometry will allow experimental detection and quantification of horizontal gene transfer events in multidrug-resistant A. baumannii.

Recombinant production of A1S_0222 from Acinetobacter baumannii ATCC 17978 and confirmation of its DNA-(adenine N6)-methyltransferase activity.

Acinetobacter baumannii appears as an often multidrug-resistant nosocomial pathogen in hospitals worldwide. Its remarkable persistence in the hospital environment is probably due to intrinsic and acquired resistance to disinfectants and antibiotics, tolerance to desiccation stress, capability to form biofilms, and is possibly facilitated by surface-associated motility. Our attempts to elucidate surface-associated motility in A. baumannii revealed a mutant inactivated in a putative DNA-(adenine N6)-methyltransferase, designated A1S_0222 in strain ATCC 17978. We recombinantly produced A1S_0222 as a glutathione S-transferase (GST) fusion protein and purified it to near homogeneity through a combination of GST affinity chromatography, cation exchange chromatography and PD-10 desalting column. Furthermore we demonstrate A1S_0222-dependent adenine methylation at a GAATTC site. We propose the name AamA (Acinetobacteradenine methyltransferase A) in addition to the formal names M.AbaBGORF222P/M.Aba17978ORF8565P. Small angle X-ray scattering (SAXS) revealed that the protein is monomeric and has an extended and likely two-domain shape in solution.

Comamonas aquatilis sp. nov., isolated from a garden pond.

A beige-pigmented bacterial strain, SB30-Chr27-3T, isolated from a garden pond, was studied for its taxonomic position. Cells of the isolate were rod-shaped and stained Gram-negative. A comparison of the 16S rRNA gene sequence with the sequences of the type strains of the most closely related species showed that the strain belongs to the genus Comamonas and showed highest sequence similarities to the type strains of Comamonas jiangduensis (97.5 %), Comamonas aquatica (97.4 %) and Comamonas phosphati (97.3 %). The 16S rRNA gene sequence similarities to all other Comamonas species were below 97.0 %. The fatty acid profile of strain SB30-Chr27-3T consisted of the major fatty acids C16 : 0, C15 : 0iso 2-OH/ C16 : 1ω7c, C18 : 1ω7c/C18 : 1ω9c and, in a minor amount, C10 : 0 3-OH. Major compounds in the polar lipid profile were phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine and diphosphatidylglycerol. The quinone system was exclusively composed of ubiquinone Q-8. The polyamine pattern contained the major compounds putrescine, cadaverine and 2-hydroxyputrescine. These data and the differentiating biochemical properties indicated that isolate SB30-CHR27-3T represents a novel species of the genus Comamonas, for which we propose the name >Comamonas aquatilis sp. nov. with the type strain SB30-Chr27-3T (=CIP 111491T=CCM 8815T).

Relatedness of wildlife and livestock avian isolates of the nosocomial pathogen Acinetobacter baumannii to lineages spread in hospitals worldwide.

The natural habitats and potential reservoirs of the nosocomial pathogen Acinetobacter baumannii are poorly defined. Here, we put forth and tested the hypothesis of avian reservoirs of A. baumannii. We screened tracheal and rectal swab samples from livestock (chicken, geese) and wild birds (white stork nestlings) and isolated A. baumannii from 3% of sampled chicken (n = 220), 8% of geese (n = 40) and 25% of white stork nestlings (n = 661). Virulence of selected avian A. baumannii isolates was comparable to that of clinical isolates in the Galleria mellonella infection model. Whole genome sequencing revealed the close relationship of an antibiotic-susceptible chicken isolate from Germany with a multidrug-resistant human clinical isolate from China and additional linkages between livestock isolates and human clinical isolates related to international clonal lineages. Moreover, we identified stork isolates related to human clinical isolates from the United States. Multilocus sequence typing disclosed further kinship between avian and human isolates. Avian isolates do not form a distinct clade within the phylogeny of A. baumannii, instead they diverge into different lineages. Further, we provide evidence that A. baumannii is constantly present in the habitats occupied by storks. Collectively, our study suggests A. baumannii could be a zoonotic organism that may disseminate into livestock.

Acinetobacter equi sp. nov., isolated from horse faeces.

The taxonomic position of five strains isolated from horse faeces, and which shared identical 16S rRNA gene sequences, were studied. Cells of all isolates are Gram-stain-negative, obligately aerobic and have a rod-shaped appearance. The strains show highest 16S rRNA gene sequence similarities to Acinetobacter lwoffii (98.3 %), Acinetobacter haemolyticus (98.0 %), Acienetobacter johnsonii (97.9 %) and Acinetobacter brisouii (97.9 %). Whole-genome sequencing of strain 114T and phylogeny reconstruction based on a core set of 1061 Acinetobacter genes indicated that A. bouvetii CIP 107468T was the closest relative among species of the genus Acinetobacter for which whole genome sequences are available. The genomic DNA G+C content of strain 114T is 34.9 mol%, which is lower than any other value reported for the genus Acinetobacter. The predominant polyamine is 1,3-diaminopropane, which is typical for the genus Acinetobacter. The most abundant fatty acids are C16 : 1ω7c and/or iso-C15 : 0 2-OH (36 %) and C16 : 0 (28 %). The proportion of C18 : 1ω9c (7 %) is distinctively low compared to most species of the genus. The major ubiquinone of strain 114T is Q-9. Microscopic studies revealed the presence of pili and the absence of flagella. The capability of all five strains to utilize l-arabinose and gentisate as well as their lack of growth at temperatures of 41 °C and above provide sufficient criteria to distinguish the isolates from all species of the genus Acinetobacter with validly published names. Based on these combined data, the five isolates represent a novel species of the genus Acinetobacter, for which the name Acinetobacter equi sp. nov. is proposed. The type strain is 114T ( = DSM 27228T = CCUG 65204T).

Analysis of Endothelial Adherence of Bartonella henselae and Acinetobacter baumannii Using a Dynamic Human Ex Vivo Infection Model.

Bacterial adherence determines the virulence of many human-pathogenic bacteria. Experimental approaches elucidating this early infection event in greater detail have been performed using mainly methods of cellular microbiology. However, in vitro infections of cell monolayers reflect the in vivo situation only partially, and animal infection models are not available for many human-pathogenic bacteria. Therefore, ex vivo infection of human organs might represent an attractive method to overcome these limitations. We infected whole human umbilical cords ex vivo with Bartonella henselae or Acinetobacter baumannii under dynamic flow conditions mimicking the in vivo infection situation of human endothelium. For this purpose, methods for quantifying endothelium-adherent wild-type and trimeric autotransporter adhesin (TAA)-deficient bacteria were set up. Data revealed that (i) A. baumannii binds in a TAA-dependent manner to endothelial cells, (ii) this organ infection model led to highly reproducible adherence rates, and furthermore, (iii) this model allowed to dissect the biological function of TAAs in the natural course of human infections. These findings indicate that infection models using ex vivo human tissue samples ("organ microbiology") might be a valuable tool in analyzing bacterial pathogenicity with the capacity to replace animal infection models at least partially.

Psychrobacter ciconiae sp. nov., isolated from white storks (Ciconia ciconia).

Five beige bacterial strains (176/10(T), 178/10, 182/10, 185/7 and 193/8) were isolated from white storks in Poland and found to share identical 16S rRNA gene sequences; they were also investigated in a polyphasic taxonomic study. The cells of all isolates were rod-shaped and Gram-stain-negative. A comparison of the 16S rRNA gene sequences of these organisms with the sequences of the type strains of the most closely related species of the genus Psychrobacter showed highest sequence similarities to the type strains of Psychrobacter pulmonis and Psychrobacter faecalis (both 97.1 %). The 16S rRNA gene sequence similarities to all other species of the genus Psychrobacter were below 96.3 %. All five isolates showed an identical profile of physiological reactions and almost identical fatty acid profiles consisting of mainly C18 : 1ω9c, but also C12 : 0 3-OH as a characteristic hydroxylated fatty acid. A quinone system with mainly ubiquinone Q-8 was detected and the polar lipid profile of the type strain, 176/10(T), was mainly composed of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine and diphosphatidylglycerol, plus some hitherto uncharacterized phospholipids and one aminolipid. The major polyamines were spermidine and putrescine. DNA-DNA hybridizations between 176/10(T) and the type strains of P. pulmonis and P. faecialis resulted in relatedness values below 70 %. These results indicate that the strains represent a novel species, for which the name Psychrobacter ciconiae sp. nov. (type strain 176/10(T) = CIP 110777(T) = LMG 28175(T) = CCM 8519(T)) is proposed.

Gemmobacter intermedius sp. nov., isolated from a white stork (Ciconia ciconia).

A cream-coloured, Gram-stain-negative, aerobic, non-motile, rod- to irregular shaped bacterium, strain 119/4(T), was isolated from a choana swab of a white stork nestling on sheep blood agar. 16S rRNA gene sequence analysis and subsequent comparisons showed that it was a member of the family Rhodobacteraceae, showing 94.9 % similarity to the type strain of Gemmobacter tilapiae and 94.6 % similarity to that of Gemmobacter nectariphilus, but also similarly low sequence similarity to the type strains of Rhodobacter viridis (94.8 %), Rhodobacter veldkampii (94.6 %) and Paenirhodobacter enshiensis (94.6 %). Reconstruction of phylogenetic trees showed that strain 119/4(T) clustered close to species of the genus Gemmobacter. The quinone system contained high amounts of ubiquinone Q-10 with traces of Q-8, Q-9 and Q-11, and the fatty acid profile consisted mainly of C18 : 1ω7c, C16 : 1ω7c/iso-C15 : 0 2-OH and C10 : 0 3-OH. The predominant polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phoshatidylglycerol and phosphatidylcholine. Major polyamines were putrescine and spermidine. On the basis of 16S rRNA gene sequence analysis and chemotaxonomic and physiological data, strain 119/4(T) represents a novel species of the genus Gemmobacter, for which the name Gemmobacter intermedius sp. nov. is proposed. The type strain is 119/4(T) ( = CIP 110795(T) = LMG 28215(T) = CCM 8510(T)).

Description of Corynebacterium trachiae sp. nov., isolated from a white stork (Ciconia ciconia).

A Gram-stain-positive bacterial isolate, strain 280/10(T) was isolated from the trachea of a white stork. The isolate was morphologically rod- to coccoid-shaped, non-motile and showed no oxidase activity. The highest 16S rRNA gene sequence similarity was found to the type strain of Corynebacterium ciconiae (97.3 % similarity) as the nearest relative of strain 280/10(T), all other 16S rRNA gene sequence similarities to type strains of species of the genus Corynebacterium were below 94.2 %. Strain 280/10(T) had a quinone system consisting predominantly of menaquinone MK-8(H2), minor quantities of MK-9(H2) and small amounts of MK-8. The diamino acid of the peptidoglycan was meso-diaminopimelic acid. The major compounds in the polar lipid profiles were diphosphatidylglycerol, phoshatidylglycerol, phosphatidylinositol and an acidic glycolipid. Two phosphatidylinositol-mannosides and several unidentified lipids were also present. The strain contained corynemycolic acids, while only small amounts of cellular polyamines were detected. Spermidine and spermine were predominant in the polyamine pattern of 280/10(T) and putrescine was present in moderate amounts. In the fatty acid profile C18 : 1ω9c, and C16 : 0 were predominant. The strain was distinguishable from C. ciconiae, which is the most closely related species. In conclusion, strain 280/10(T) is proposed to represent a novel species of the genus Corynebacterium with the name Corynebacterium trachiae sp. nov. and the type strain 280/10(T) ( = CIP 110796(T) = LMG 28214(T)).

Corynebacterium pelargi sp. nov., isolated from the trachea of white stork nestlings.

A Gram-stain-positive, pleomorphic, oxidase-negative, non-motile isolate from the trachea of a white stork from Poland, designated strain 136/3(T), was subjected to a comprehensive taxonomic investigation. A comparative analysis of the 16S rRNA gene sequence showed highest similarities to Corynebacterium mustelae , Corynebacterium pseudotuberculosis , Corynebacterium vitaeruminis and Corynebacterium ulcerans (96.0-96.3%). The quinone system consisted of major amounts of MK-8(H2), minor amounts of MK-9(H2) and traces of MK-8 and MK-9. The polar lipid profile of strain 136/3(T) contained phosphatidylinositol and phosphatidylinositol-mannoside as major lipids and phosphatidylglycerol and an acidic glycolipid in moderate amounts. In addition small amounts of diphosphatidylglycerol, a phospholipid, an aminolipid and two lipids of unknown group affiliation were found. The polyamine pattern was composed of the major components spermidine and spermine. Putrescine, 1,3-diaminopropane, cadaverine, sym-homospermidine and tyramine were found in minor or trace amounts. The diamino acid of the peptidoglycan was meso-diaminopimelic acid. In the fatty acid profile straight-chain, saturated and mono-unsaturated fatty acids predominated (C(18 : 1)ω9c, C(16 : 1)ω7c, C16 : 0, C(18  : 0)). Corynemycolic acids were detected. Physiological traits as well as unique traits of the polar lipid profile and the fatty acid pattern distinguished strain 136/3(T) from the most closely related species. All these results indicate that strain 136/3(T) represents a novel species of the genus Corynebacterium for which we propose the name Corynebacterium pelargi sp. nov. The type strain is 136/3(T) ( =CIP 110778(T) =CCM 8517(T) =LMG 28174(T)).

Chryseobacterium gallinarum sp. nov., isolated from a chicken, and Chryseobacterium contaminans sp. nov., isolated as a contaminant from a rhizosphere sample.

Two yellow-pigmented bacterial strains (100(T) and C26(T)), showing 98.4 % 16S rRNA gene sequence similarity to each other and isolated from a chicken in Germany and as a contaminant from an agar plate of a rhizosphere sample in Alabama, were studied by using a polyphasic taxonomic approach. Cells of both isolates were rod-shaped and stained Gram-negative. A comparison of the 16S rRNA gene sequences of the two organisms with the sequences of the type strains of the most closely related species of the genus Chryseobacterium showed the highest sequence similarities of strains 100(T) and C26(T) to the type strains of Chryseobacterium joostei (respectively 97.5 and 98.2 %), C. viscerum (96.6, 97.8 %), C. gleum (97.1, 97.7 %), C. arthrosphaerae (97.3%, 97.7 %), C. indologenes (97.2, 97.7 %), C. tructae (96.6, 97.6 %), C. jejuense (97.0, 97.6 %) and C. oncorhynchi (96.3, 97.5 %); 16S rRNA gene sequence similarities to members of all other species of the genus Chryseobacterium were below 97.5 %. The fatty acid profiles of both strains consisted of the major fatty acids iso-C15 : 0, summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1ω7c), iso-C17 : 1ω9c and iso-C17 : 0 3-OH, but also showed slight differences (absence or presence of C16 : 0 3-OH and iso-C15 : 1 F). DNA-DNA hybridizations between the two strains and between the novel strains and the type strains of C. joostei, C. indologenes, C. jejuense, C. tructae and C. viscerum resulted in relatedness values clearly below 70 %. These DNA-DNA hybridization results and the differentiating biochemical and chemotaxonomic properties showed that both strains 100(T) and C26(T) represent novel species, for which the names Chryseobacterium gallinarum sp. nov. (type strain 100(T) = LMG 27808(T) = CCM 8493(T)) and Chryseobacterium contaminans sp. nov. (type strain C26(T) = LMG 27810(T) = CCM 8492(T)) are proposed.