Abstract
T was isolated from the hindgut of Tipula abdominalis larvae as described by Cook et al. (2007) . T. abdominalis is an aquatic crane fly, larvae of which are primary shredders of leaf litter in small, riparian streams. The hindgut of T. abdominalis larvae hosts a dense and diverse bacterial community (Klug & Kotarski, 1980) , which is suggested to facilitate digestion of their lignocellulosic diet (Lawson & Klug, 1989) .
T was maintained as 40 % (w/v) glycerol suspensions at 220 u C. Culture for biochemical and molecular studies was obtained by cultivation on trypticase soy agar (TSA; Difco) or in trypticase soy broth (TSB; Difco) at 28 u C for 48 h. Cultures were incubated at 4, 10, 22, 28, 30, 37 and 45 u C to determine the range and optimum temperature for growth. At 28 u C, growth was tested at pH 6-12 and in the presence of NaCl concentrations of 0.5-9 % to determine the pH and NaCl optima and range. Colony morphology was observed on TSA after 48 h growth at 28 u C. Gram staining was performed and standard physiological tests were performed with API NE, API Staph, API Strep and API Coryne test kits (bioMérieux). For phase-contrast microscopy observation, cells were viewed at 6100 magnification with a Leica SP2 upright microscope (Leica Microsystems Inc.) and images were captured with a Zeiss AxioCam (Carl Zeiss MicroImaging, Inc.) at the Center for Advanced Ultrastructure Research at the University of Georgia. A pixel to micrometre ratio was calculated via imaging software and this ratio was used to determine cell size.
T stained Gram-variable, but was Gram-type positive. It was aerobic, grew optimally at 28 u C and was able to grow at 4-30 u C. Although limited growth did occur at 4 u C, this was not observed until 672 h (4 weeks) of incubation. Irregular rod-shaped cells (0.6-3.460.4-0.8 mm) were observed, but spores were not found. Small, smooth, yellow colonies formed on TSA. Detailed biochemical and physiological characteristics of the strain are given in the genus and species descriptions below.
Analyses of cell-wall sugars, menaquinones, amino acids and acyl type were performed by the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under the direction of Dr Peter Schumann according to recognized methods (Groth et al., 1996; Schleifer, 1985; Schleifer & Kandler, 1972; Staneck & Roberts, 1974; Uchida et al., 1999) . Total cellular fatty acids were analysed by using the MIDI-FAME procedure essentially as described by Haack et al. (1994) ; gas chromatographs were compared
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain 44C3
T is AY372075.
A minimum-evolution phylogenetic dendrogram based on 16S rRNA gene sequence similarity is available as supplementary material with the online version of this paper.

within the family Microbacteriaceae, for which the name Klugiella xanthotipulae gen. nov., sp. nov. is proposed. The type strain of Klugiella xanthotipulae is 44C3 T (5DSM 18031 T 5ATCC BAA-
T
).
Strain 44C3
T was isolated from the hindgut of Tipula abdominalis larvae as described by Cook et al. (2007) . T. abdominalis is an aquatic crane fly, larvae of which are primary shredders of leaf litter in small, riparian streams. The hindgut of T. abdominalis larvae hosts a dense and diverse bacterial community (Klug & Kotarski, 1980) , which is suggested to facilitate digestion of their lignocellulosic diet (Lawson & Klug, 1989) .
T was maintained as 40 % (w/v) glycerol suspensions at 220 u C. Culture for biochemical and molecular studies was obtained by cultivation on trypticase soy agar (TSA; Difco) or in trypticase soy broth (TSB; Difco) at 28 u C for 48 h. Cultures were incubated at 4, 10, 22, 28, 30, 37 and 45 u C to determine the range and optimum temperature for growth. At 28 u C, growth was tested at pH 6-12 and in the presence of NaCl concentrations of 0.5-9 % to determine the pH and NaCl optima and range. Colony morphology was observed on TSA after 48 h growth at 28 u C. Gram staining was performed and standard physiological tests were performed with API NE, API Staph, API Strep and API Coryne test kits (bioMérieux). For phase-contrast microscopy observation, cells were viewed at 6100 magnification with a Leica SP2 upright microscope (Leica Microsystems Inc.) and images were captured with a Zeiss AxioCam (Carl Zeiss MicroImaging, Inc.) at the Center for Advanced Ultrastructure Research at the University of Georgia. A pixel to micrometre ratio was calculated via imaging software and this ratio was used to determine cell size.
T stained Gram-variable, but was Gram-type positive. It was aerobic, grew optimally at 28 u C and was able to grow at 4-30 u C. Although limited growth did occur at 4 u C, this was not observed until 672 h (4 weeks) of incubation. Irregular rod-shaped cells (0.6-3.460.4-0.8 mm) were observed, but spores were not found. Small, smooth, yellow colonies formed on TSA. Detailed biochemical and physiological characteristics of the strain are given in the genus and species descriptions below.
Analyses of cell-wall sugars, menaquinones, amino acids and acyl type were performed by the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under the direction of Dr Peter Schumann according to recognized methods (Groth et al., 1996; Schleifer, 1985; Schleifer & Kandler, 1972; Staneck & Roberts, 1974; Uchida et al., 1999) . Total cellular fatty acids were analysed by using the MIDI-FAME procedure essentially as described by Haack et al. (1994) ; gas chromatographs were compared against profiles generated with authenticated standards and archived profiles from known cultures grown under standard conditions by using the MIDI Microbial Identification software (MIDI Inc.).
Rhamnose was the only cell-wall sugar. The major menaquinones were MK-12 and MK-11. The fatty acid profile contained ai-C 15 : 0 (54.71 %), ai-C 17 : 0 (18.28 %), i-C 16 : 0 (17.92 %), i-C 15 : 0 (1.37 %) and i-C 14 : 0 (1.03 %). No glycosyl residues were found in the peptidoglycan, and thus the peptidoglycan was of the acetyl type. Analysis of the cell-wall amino acids revealed that the peptidoglycan of strain 44C3
T contained alanine, glycine, glutamate and lysine at a molar ratio of 1.6 : 0.9 : 1.0 : 1.0. No other amino acids found in some B-type peptidoglycans (including diaminobutyric acid, ornithine, homoserine and hydroxyglutamic acid) could be detected. As usual for B-type peptidoglycans, the peptide Gly-D-Glu was detected in the partial hydrolysate. The peptide D-Ala-Ala was found, confirming alanine as the N terminus of the interpeptide bridge. Three additional peptides were found, which probably comprised lysine and alanine residues. Although these data were not sufficient to propose a definite structure, they do not concur with published peptidoglycan structures, and support the conclusion that the peptidoglycan of strain 44C3
T represents a novel B-type.
Sequencing of the 16S rRNA gene was performed at MIDI Laboratories. Putative strain identity was determined by searching catalogued sequences in GenBank (Benson et al., 2005) by using the BLAST tool (Altschul et al., 1990) . Sequence alignments among strain 44C3 T and the type strains of the most closely related actinobacteria were created by using the CLUSTAL_X program (Thompson et al., 1997) , and these alignments were edited in GeneDoc (Nicholas et al., 1997) . Distances were calculated by using the Jukes-Cantor algorithm (Jukes & Cantor, 1969) , and branching order was calculated with the neighbour-joining method (Saitou & Nei, 1987) . A phylogenetic tree was constructed by using the program MEGA 3.1, which calculates bootstrap values internally (Kumar et al., 2004) . To confirm the phylogenetic position of strain 44C3 T , a minimum-evolution algorithm analysis was also performed with the MEGA 3.1 program (see Supplementary  Fig. S1 in IJSEM Online). Extraction of genomic DNA was performed by using French pressure cell lysis (Thermo Spectronic), followed by purification via chromatography on hydroxyapatite as described by Cashion et al. (1977) . The DNA G+C content was determined according to Mesbah et al. (1989) , and was confirmed by the DSMZ under the direction of P. Schumann according to standard methods (Cashion et al., 1977; Mesbah et al., 1989; Tamaoka & Komagata, 1984; Visuvanathan et al., 1989) .
The nearest phylogenetic neighbours of strain 44C3
T , as determined based on analysis of its 16S rRNA gene sequence (1501 bp), were distantly related members of the family Microbacteriaceae (similarities ranging from 92.5 to 96.5 %). Strain 44C3
T formed a distinct subclade within the family, and showed highest 16S rRNA gene sequence similarity to Cryobacterium psychrotolerans 0549 T (96.5 %) (Fig. 1) . The G+C content of the genomic DNA of strain 44C3 T was 60.9 mol%. Bar, 1 % sequence divergence. A minimumevolution tree is available as Supplementary  Fig. S1 .
Genera of the family Microbacteriaceae contain rhamnose as well as one or more other sugars in the cell wall (see references in Table 1 ), whereas rhamnose was the only sugar detected in strain 44C3 T . Strain 44C3 T was similar to members of the genera Mycetocola, Frigoribacterium and Microcella in having lysine as a cell-wall diamino acid, but differed in its major menaquinones, major fatty acids and DNA G+C content. In terms of the quinone system, members of the genus Agrococcus (Groth et al., 1996) have menaquinones similar to those of strain 44C3
T , but they differ in other chemotaxonomic characteristics, including peptidoglycan amino acids, major fatty acid composition and DNA G+C content (Table 1) .
Chemotaxonomic characteristics that differentiate strain 44C3
T from representatives of its nearest phylogenetic neighbours detected based on 16S rRNA gene sequence analysis are reported in Table 1 . It is evident from the genotypic and phenotypic data presented that strain 44C3 T represents a novel species of a new genus within the family Microbacteriaceae, for which the name Klugiella xanthotipulae gen. nov., sp. nov. is proposed. Klugiella can be distinguished from other genera of the Microbacteriaceae based on its major menaquinones (MK-12 and MK-11) and cell-wall diamino acid (lysine). Some species of the genus Microbacterium (Table 1) have the above characteristics, but Klugiella can be differentiated from them by having rhamnose as the only detectable cell-wall sugar and having a lower DNA G+C content, of approximately 61 mol%.
Description of Klugiella gen. nov. Klugiella (Klu.gi.el9la. N.L. fem. n. Klugiella named after Michael J. Klug, an American entomologist/microbiologist who, along with S. Kotarski, first described the microbial community of the Tipula abdominalis larval gut, from which strain 44C3 T was isolated).
Gram-type positive, Gram-reaction variable, mesophilic and aerobic. Cells are non-motile, non-spore-forming, irregular rods (0.6-3.460.4-0.8 mm). The peptidoglycan type is B, lysine is the diamino acid of the peptidoglycan and alanine is the N terminus of the interpeptide bridge. The major menaquinones are MK-12 and MK-11. The major fatty acids are ai-C 15 : 0 , ai-C 17 : 0 and i-C 16 : 0 . The G+C content of the genomic DNA is about 61 mol%. 16S rRNA gene sequence similarity indicates membership of the family Microbacteriaceae. The type species is Klugiella xanthotipulae.
Description of Klugiella xanthotipulae sp. nov.
Klugiella xanthotipulae (xan9tho.ti9pu.lae. Gr. adj. xanthos yellow; N.L. fem. gen. n. tipulae of Tipula, a zoological genus name; N.L. fem. gen. n. xanthotipulae yellow from Tipula, referring to the isolation of a yellow-colonyforming organism from Tipula abdominalis).
Colonies are convex, circular and yellow. Growth occurs at 4-30 u C with an optimum at 28 u C and at a pH range of 6-11 with an optimum at pH 8. Can grow in the presence of 1 % (w/v) NaCl but not 3 %. Catalase-positive and oxidasenegative. Positive for pyrazinamidase, b-glucuronidase, bgalactosidase and a-glucosidase. Negative for a-galactosidase, arginine dihydrolase, leucine arylamidase, pyrrolidonyl arylamidase, alkaline phosphatase, N-acetyl-bglucosaminidase and urease, indole production, acetoin production, hydrolysis of gelatin and reduction of nitrates. Negative for acid production from L-arabinose, lactose, ribose and sorbitol. Acid is produced from fructose, The type strain, 44C3 T (5DSM 18031 T 5ATCC BAA-1524 T ), was isolated from the hindgut of Tipula abdominalis larvae collected in Michigan, USA.

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