Characterization of Xanthomonas campestris Pathovars by rRNA Gene Restriction Patterns

Genomic DNA of 191 strains of the family Pseudomonadaceae, including 187 strains of the genus Xanthomonas, was cleaved by EcoRI endonuclease. After hybridization of Southern transfer blots with 2-acetylamino-fluorene-labelled Escherichia coli 16+23S rRNA probe, 27 different patterns were obtained. The strains are clearly distinguishable at the genus, species, and pathovar levels. The variability of the rRNA gene restriction patterns was determined for four pathovars of Xanthomonas campestris species. The 16 strains of X. campestris pv. begoniae analyzed gave only one pattern. The variability of rRNA gene restriction patterns of X. campestris pv. manihotis strains could be related to ecotypes. In contrast, the variability of patterns observed for X. campestris pv. malvacearum was not correlated with pathogenicity or with the geographical origins of the strains. The highest degree of variability of DNA fingerprints was observed within X. campestris pv. dieffenbachiae, which is pathogenic to several hosts of the Araceae family. In this case, variability was related to both host plant and pathogenicity.     

Estimation of 16S rRNA gene copy number in several probiotic Lactobacillus strains isolated from the gastrointestinal tract of chicken

The copy numbers of 16S rRNA genes in 12 probiotic Lactobacillus strains of poultry origin were analyzed. Genomic DNA of the strains was digested with restriction endonucleases that do not cut within the 16S rRNA gene of the strains. This was followed by Southern hybridization with a biotinylated probe complementary to the 16S rRNA gene. The copy number of the 16S rRNA gene within a Lactobacillus species was found to be conserved. From the hybridization results, Lactobacillus salivarius I 24 was estimated to have seven copies of the 16S rRNA gene, Lactobacillus panis C 17 to have five copies and Lactobacillus gallinarum strains I 16 and I 26 four copies. The 16S rRNA gene copy numbers of L. gallinarum and L. panis reported in the present study are the first record. Lactobacillus brevis strains I 12, I 23, I 25, I 211, I 218 and Lactobacillus reuteri strains C 1, C 10, C 16 were estimated to have at least four copies of the 16S rRNA gene. In addition, distinct rRNA restriction patterns which could discriminate the strains of L. reuteri and L. gallinarum were also detected. Information on 16S rRNA gene copy number is important for physiological, evolutionary and population studies of the bacteria.     

Toward an International Standard for PCR-Based Detection of Food-Borne Thermotolerant Campylobacters: Assay Development and Analytical Validation

As part of a European research project (FOOD-PCR), we developed a standardized and robust PCR detection assay specific for the three most frequently reported food-borne pathogenic Campylobacter species, C. jejuni, C. coli, and C. lari. Fifteen published and unpublished PCR primers targeting the 16S rRNA gene were tested in all possible pairwise combinations, as well as two published primers targeting the 23S rRNA gene. A panel of 150 strains including target and nontarget strains was used in an in-house validation. Only one primer pair, OT1559 plus 18-1, was found to be selective. The inclusivity and exclusivity were 100 and 97%, respectively. In an attempt to find a thermostable DNA polymerase more resistant than Taq to PCR inhibitors present in chicken samples, three DNA polymerases were evaluated. The DNA polymerase Tth was not inhibited at a concentration of 2% (vol/vol) chicken carcass rinse, unlike both Taq DNA polymerase and DyNAzyme. Based on these results, Tth was selected as the most suitable enzyme for the assay. The standardized PCR test described shows potential for use in large-scale screening programs for food-borne Campylobacter species under the assay conditions specified.     

Genista tinctoria microsymbionts from Poland are new members of Bradyrhizobium japonicum bv. genistearum

The phylogeny and taxonomic position of slow-growing Genista tinctoria rhizobia from Poland, Ukraine and England were estimated by comparative 16S rDNA, atpD, and dnaK sequence analyses, PCR-RFLP of 16S rDNA, DNA G + C content, and DNA–DNA hybridization. Each core gene studied placed the G. tinctoria rhizobia in the genus Bradyrhizobium cluster with unequivocal bootstrap support. G. tinctoria symbionts and bradyrhizobial strains shared 96–99% similarity in 16S rDNA sequences. Their similarity for atpD and dnaK sequences was 93–99% and 89–99%, respectively. These data clearly showed that G. tinctoria rhizobia belonged to the genus Bradyrhizobium. 16S rDNA sequence analysis was in good agreement with the results of the PCR-RFLP of the 16S rRNA gene. Although the tested strains formed separate lineages to the reference bradyrhizobia their RFLP 16S rDNA patterns were quite similar. The genomic DNA G + C content of three G. tinctoria rhizobia was in the range from 60.64 to 62.83 mol%. Data for species identification were obtained from DNA–DNA hybridization experiments. G. tinctoria microsymbionts from Poland were classified within Bradyrhizobium japonicum genomospecies based on 56–82% DNA–DNA similarity.     

Campylobacter insulaenigrae Isolates from Northern Elephant Seals (Mirounga angustirostris) in California

There are only two reports in the literature demonstrating the presence of Campylobacter spp. in marine mammals. One report describes the isolation of a new species, Campylobacter insulaenigrae sp. nov., from three harbor seals (Phoca vitulina) and a harbor porpoise (Phocoena phocoena) in Scotland, and the other describes the isolation of Campylobacter jejuni, Campylobacter lari, and an unknown Campylobacter species from northern elephant seals (Mirounga angustirostris) in California. In this study, 72 presumptive C. lari and unknown Campylobacter species strains were characterized using standard phenotypic methods, 16S rRNA PCR, and multilocus sequence typing (MLST). Phenotypic characterization of these isolates showed them to be variable in their ability to grow either at 42°C or on agar containing 1% glycine and in their sensitivity to nalidixic acid and cephalothin. Based on both 16S rRNA PCR and MLST, all but 1 of the 72 isolates were C. insulaenigrae, with one isolate being similar to but distinct from both Campylobacter upsaliensis and Campylobacter helveticus. Phylogenetic analysis identified two C. insulaenigrae clades: the primary clade, containing exclusively California strains, and a secondary clade, containing some California strains and all of the original Scottish strains. This study demonstrates the inability of phenotypic characterization to correctly identify all Campylobacter species and emphasizes the importance of molecular characterization via 16S rRNA sequence analysis or MLST for the identification of Campylobacter isolates from marine mammals.     

Fingerprinting of prokaryotic 16S rRNA genes using oligodeoxyribonucleotide microarrays and virtual hybridization

An oligonucleotide microarray hybridization system to differentiate microbial species was designed and tested. Seven microbial species were studied, including one Bacillus and six Pseudomonas strains. DNA sequences near the 5′ end of 16S rRNA genes were aligned and two contiguous regions of high variability, flanked by highly conserved sequences, were found. The conserved sequences were used to design PCR primers which efficiently amplified these polymorphic regions from all seven species. The amplicon sequences were used to design 88 9mer hybridization probes which were arrayed onto glass slides. Single-stranded, fluorescence-tagged PCR products were hybridized to the microarrays at 15°C. The experimental results were compared with the ΔG° values for all matched and mismatched duplexes possible between the synthetic probes and the 16S target sequences of the seven test species, calculated using a ‘virtual hybridization’ software program. Although the observed hybridization patterns differed significantly from patterns predicted solely on the basis of perfect sequence matches, a unique hybridization fingerprint was obtained for each of the species, including closely related Pseudomonas species, and there was a reasonable correlation between the intensity of observed hybridization signals and the calculated ΔG° values. The results suggest that both perfect and mismatched pairings can contribute to microbial identification by hybridization fingerprinting.     

Molecular fingerprinting of isolates of the genus Peptostreptococcus using rRNA genes from Escherichia coli and P. anaerobius.

Restriction fragment length polymorphisms (RFLPs) of rRNA genes were evaluated as a tool for intra- and interspecies differentiation of Peptostreptococcus isolates. RFLPs from a collection of 20 clinical isolates and five ATCC strains representing five Peptostreptococcus spp. (P. anaerobius, P. asaccharolyticus, P. magnus, P. micros and P. prevotii) were obtained by hybridization of Southern blots of HindIII- or EcoRI-digested genomic DNA with three probes: probe A, a 0.98 kb HindIII fragment with a partial 16S rRNA gene sequence from P. anaerobius ATCC 27337; probe B, cloned Escherichia coli rrnB operon in plasmid pKK3535; and probe C, E. coli 16S and 23S rRNA. The hybridization patterns varied, but all yielded RFLPs useful for both intra- and inter-species differentiation. RFLPs of P. asaccharolyticus clinical isolates were closely related to each other and differed significantly from those of the ATCC type strains. The profiles of P. prevotii differed from those of the other four species studied, and based on the HindIII- and EcoRI-generated RFLPs, the strains in this species are more heterogeneous than the other four species studied.     

Development of a Simple and Rapid Method Based on Polymerase Chain Reaction–Based Restriction Fragment Length Polymorphism Analysis to Differentiate Helicobacter, Campylobacter, and Arcobacter Species

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of amplified DNA fragment of the 16S and 23S rRNA genes was performed on 35 Helicobacter, 24 Campylobacter, and 15 Arcobacter strains. PCR amplification generated a 1004-bp fragment of 16S rDNA and a 2.6-Kbp fragment of 23S rDNA from each strain. The amplicons were digested with DdeI and HpaII, respectively. For both assays, distinctive profiles were obtained for each genus. 23S rDNA PCR-RFLP analysis with HpaII enzyme identified Campylobacter and Helicobacter strains at the species level. Analysis of 16S rRNA gene with DdeI enzyme was not useful for the specific identification of Campylobacter and Arcobacter, although it discriminated among Helicobacter species. The PCR-RFLP technique allowed for the discrimination among these three related genus with only one restriction enzyme; therefore it can be a simple, rapid, and useful method for routine identification.     

Toxicity phenotype does not correlate with phylogeny of Cylindrospermopsis raciborskii strains

Cylindrospermopsis raciborskii is a species of freshwater, bloom-forming cyanobacterium. C. raciborskii produces toxins, including cylindrospermopsin (hepatotoxin) and saxitoxin (neurotoxin), although non toxin-producing strains are also observed. In spite of differences in toxicity, C. raciborskii strains comprise a monophyletic group, based upon 16S rRNA gene sequence identities (greater than 99%). We performed phylogenetic analyses; 16S rRNA gene and 16S-23S rRNA gene internally transcribed spacer (ITS-1) sequence comparisons, and genomic DNA restriction fragment length polymorphism (RFLP), resolved by pulsed-field gel electrophoresis (PFGE), of strains of C. raciborskii, obtained mainly from the Australian phylogeographic cluster. Our results showed no correlation between toxic phenotype and phylogenetic association in the Australian strains. Analyses of the 16S rRNA gene and the respective ITS-1 sequences (long L, and short S) showed an independent evolution of each ribosomal operon. The genes putatively involved in the cylindrospermopsin biosynthetic pathway were present in one locus and only in the hepatotoxic strains, demonstrating a common genomic organization for these genes and the absence of mutated or inactivated biosynthetic genes in the non toxic strains. In summary, our results support the hypothesis that the genes involved in toxicity may have been transferred as an island by processes of gene lateral transfer, rather than convergent evolution.     

Cloning and characterization of genes coding for ribosomal RNA inCampylobacter jejuni

The DNA fragments coding for ribosomal RNA inCampylobacter jejuni have been cloned from a genomic library ofC. jejuni constructed inEscherichia coli. Clones carrying DNA Sequences for rRNA were identified by hybridization of 5-end-labeled rRNA fromC. jejuni to colony blots of transformants from this gene library. Cloned DNA sequences homologous to each of 5S, 16S, and 23S rRNA were idenfified by hybridization of labeled plasmid DNA to Northern blots of rRNA. The gene coding for 23S rRNA was found to be located on a 5.5kb HindIII fragment, while the 5S and 16S rRNA genes were on HindIII fragments of 1.65 and 1.7 kb, respecitively. The DNA fragment containing the 16S rRNA gene was characterized by restriction endonuclease mapping, and the location of the 16S rRNA gene on this fragment was determined by hybridization of 5-end-labeled rRNA to restriction fragments and also by DNA sequence determination. It appears that the major portion of the coding region for 16S rRNA is located on the 1.7-kb HindIII fragment, while a small portion is carried on an adjacent HindIII fragment of 7.5 kb. Cloned rRNA genes fromC. jejuni were used to study the organization of the rDNA inC. jejuni and other members of the genùsCampylobacter.     

Differentiation of ruminal and human Streptococcus bovis strains by DNA homology and 16s rRNA probes

Streptococcus bovis is commonly present in the rumen, but strains of S. bovis have also occasionally been isolated from human blood or fecal samples. Studies were undertaken with 16s rRNA gene sequences and DNA hybridizations to define the genetic relationships between these two groups of strains. Ruminal strains were found to yield genomic DNA restriction endonuclease digest patterns different from human strains when either the 16s rRNA gene amplified from ruminal S. bovis strain JB1 or a conserved universal 23s rRNA fragment was used as probes. A DNA probe based on the V1 region of the 16s rRNA of S. bovis JB1 was found to hybridize to DNAs of other ruminal S. bovis strains K27FF4, 21-09-6C, five new ruminal isolates, and weak hybridization was found with DNAs from S. bovis 33317 (type strain), S. equinus 9812, and six other ruminal isolates. No hybridization occurred with strains representing different major human biotypes/homology groups (43143, 43144, 27960, V1387). All ruminal S. bovis strains had a guanosine plus cytosine DNA content of 37.4–38.8 mol% and, based on DNA-DNA genomic hybridizations, could be separated into two homology groups, one of which included S. equinus 9812 and S. bovis 33317. Both ruminal groups had less than 38% DNA homology to the human strains, indicating ruminal strains are clearly two separate species distinct from the human strains.     

Vibrio cortegadensis sp. nov., isolated from clams

A group of four strains isolated from clams (Venerupis decussata and Venerupis philippinarum) in Galicia (NW Spain) were subjected to a polyphasic characterization, based on the phenotypic characteristics, the analysis of chemotaxonomic features, the sequencing of the 16S rRNA and five housekeeping (atpA, pyrH, recA, rpoA and rpoD) genes, as well as DNA–DNA hybridization (DDH). The analysis of the phenotypic and chemotaxonomic characteristics and the results of a phylogenetic study, based on the 16S rRNA gene sequence analysis and multilocus sequence analysis, clearly indicated that these strains belong to the genus Vibrio and were allocated between the Splendidus and Anguillarum clades showing a close relationship with the type strains of Vibrio tapetis (98.8 %), Vibrio pomeroyi (98.0 %) and Vibrio crassostreae (97.9 %). DNA–DNA hybridization results confirmed that these isolates constitute a new species. The name Vibrio cortegadensis sp. nov. is proposed with C 16.17^T (=CECT 7227^T=LMG 27474^T) as the type strain.     

Identification of nine sequence types of the 16S rRNA genes of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> subsp. <Emphasis Type="Italic">jejuni</Emphasis> isolated from broilers

Background  

Campylobacter is the most commonly reported bacterial cause of enteritis in humans in the EU Member States and other industrialized countries. One significant source of infection is broilers and consumption of undercooked broiler meat. Campylobacter jejuni is the Campylobacter sp. predominantly found in infected humans and colonized broilers. Sequence analysis of the 16S rRNA gene is very useful for identification of bacteria to genus and species level. The objectives in this study were to determine the degree of intraspecific variation in the 16S rRNA genes of C. jejuni and C. coli and to determine whether the 16S rRNA sequence types correlated with genotypes generated by PFGE analysis of Sma I restricted genomic DNA of the strains.     

Yersinia spp. Identification Using Copy Diversity in the Chromosomal 16S rRNA Gene Sequence

API 20E strip test, the standard for Enterobacteriaceae identification, is not sufficient to discriminate some Yersinia species for some unstable biochemical reactions and the same biochemical profile presented in some species, e.g. Yersinia ferderiksenii and Yersinia intermedia, which need a variety of molecular biology methods as auxiliaries for identification. The 16S rRNA gene is considered a valuable tool for assigning bacterial strains to species. However, the resolution of the 16S rRNA gene may be insufficient for discrimination because of the high similarity of sequences between some species and heterogeneity within copies at the intra-genomic level. In this study, for each strain we randomly selected five 16S rRNA gene clones from 768 Yersinia strains, and collected 3,840 sequences of the 16S rRNA gene from 10 species, which were divided into 439 patterns. The similarity among the five clones of 16S rRNA gene is over 99% for most strains. Identical sequences were found in strains of different species. A phylogenetic tree was constructed using the five 16S rRNA gene sequences for each strain where the phylogenetic classifications are consistent with biochemical tests; and species that are difficult to identify by biochemical phenotype can be differentiated. Most Yersinia strains form distinct groups within each species. However Yersinia kristensenii, a heterogeneous species, clusters with some Yersinia enterocolitica and Yersinia ferderiksenii/intermedia strains, while not affecting the overall efficiency of this species classification. In conclusion, through analysis derived from integrated information from multiple 16S rRNA gene sequences, the discrimination ability of Yersinia species is improved using our method.     

Vagococcus teuberi sp. nov., isolated from the Malian artisanal sour milk fènè

Ten bacterial isolates belonging to the genus Vagococcus were obtained from Malian sour milk fènè produced from spontaneously fermented cow milk. However, these isolates could not be assigned to a species upon initial comparative 16S rRNA gene sequence analysis and were therefore further characterized. Rep-PCR fingerprinting of the isolates yielded four strain clusters represented by strains CG-21^T (=DSM 21459^T), 24CA, CM21 and 9H. Sequence identity of the 16S rRNA gene of DSM 21459^T to its closest relative species Vagococcus penaei was 97.9%. Among the four rep strain clusters, DSM 21459^T and 24CA shared highest 16S rRNA gene sequence identity of 99.6% while CM21 and 9H shared 98.6–98.8% with DSM 21459^T and V. penaei CD276^T. DSM 21459^T and 24CA were thus subjected to a polyphasic typing approach. The genome of DSM 21459^T featured a G + C content of 34.1 mol% for a 2.17-bp chromosome and a 15-kbp plasmid. Average nucleotide identity (ANI) of DSM 21459^T to Vagococcus fluvialis bH819, V. penaei CD276^T were 72.88%, 72.63%, respectively. DNA–DNA hybridization (DDH) similarities of strain DSM 21459^T to other Vagococcus species were <42.0%. ANI and DDH findings strongly supported the 16S rRNA gene phylogenetic tree delineations. The fatty acid patterns of DSM 21459^T was palmitic acid (C _16:0, 24.5%), oleic acid (C _18:1-ω9c, 32.8%), stearic acid (C _18:0, 18.9%). General physiological characterization of DSM 21459^T and 24CA were consistent with those of the genus Vagococcus. Strain DSM 21459^T and further strains are therefore considered to belong to a novel species, for which the nomenclature Vagococcus teuberi sp. nov. is proposed. The type strain is named CG-21^T (=DSM 21459^T and LMG 24695^T).     

Rapid Detection, Identification, and Enumeration of Escherichia coli Cells in Municipal Water by Chemiluminescent In Situ Hybridization

A new chemiluminescent in situ hybridization (CISH) method provides simultaneous detection, identification, and enumeration of culturable Escherichia coli cells in 100 ml of municipal water within one working day. Following filtration and 5 h of growth on tryptic soy agar at 35°C, individual microcolonies of E. coli were detected directly on a 47-mm-diameter membrane filter using soybean peroxidase-labeled peptide nucleic acid (PNA) probes targeting a species-specific sequence in E. coli 16S rRNA. Within each microcolony, hybridized, peroxidase-labeled PNA probe and chemiluminescent substrate generated light which was subsequently captured on film. Thus, each spot of light represented one microcolony of E. coli. Following probe selection based on 16S ribosomal DNA (rDNA) sequence alignments and sample matrix interference, the sensitivity and specificity of the probe Eco16S07C were determined by dot hybridization to RNA of eight bacterial species. Only the rRNA of E. coli and Pseudomonas aeruginosa were detected by Eco16S07C with the latter mismatch hybridization being eliminated by a PNA blocker probe targeting P. aeruginosa 16S rRNA. The sensitivity and specificity for the detection of E. coli by PNA CISH were then determined using 8 E. coli strains and 17 other bacterial species, including closely related species. No bacterial strains other than E. coli and Shigella spp. were detected, which is in accordance with 16S rDNA sequence information. Furthermore, the enumeration of microcolonies of E. coli represented by spots of light correlated 92 to 95% with visible colonies following overnight incubation. PNA CISH employs traditional membrane filtration and culturing techniques while providing the added sensitivity and specificity of PNA probes in order to yield faster and more definitive results.     

Restriction fragment length polymorphisms of 16S rRNA genes in the differentiation of fast-growing mycobacterial species

Abstract DNA from several species of fast growing mycobacteria displayed a characteristic restriction fragment length polymorphism (RFLP) pattern when hybridizated to a Mycobacterium fortuitum 16S rRNA gene fragment. The resulting patterns were identical when comparing different strains belonging to the same species. The RFLP results were consistent with those obtained by DNA-DNA hybridization studies. Using this approach, we have been able to identify the number of copies for 16S rRNA genes in several fast-growing mycobacteria.     

Dissemination of Fluoroquinolone-Resistant Campylobacter spp. within an Integrated Commercial Poultry Production System

While characterizing the intestinal bacterial community of broiler chickens, we detected -proteobacterial DNA in the ilea of 3-day-old commercial broiler chicks (J. Lu, U. Idris, B. Harmon, C. Hofacre, J. J. Maurer, and M. D. Lee, Appl. Environ. Microbiol. 69:6816-6824, 2003). The sequences exhibited high levels of similarity to Campylobacter jejuni and Campylobacter coli sequences, suggesting that chickens can carry Campylobacter at a very young age. Campylobacter sp. was detected by PCR in all samples collected from the ilea of chicks that were 3 to 49 days old; however, it was detected only in the cecal contents of chickens that were at least 21 days old. In order to determine whether the presence of Campylobacter DNA in young chicks was due to ingestion of the bacteria in food or water, we obtained commercial broiler hatching eggs, which were incubated in a research facility until the chicks hatched. DNA sequencing of the amplicons resulting from Campylobacter-specific 16S PCR performed with the ileal, cecal, and yolk contents of the day-of-hatching chicks revealed that Campylobacter DNA was present before the chicks consumed food or water. The 16S rRNA sequences exhibited 99% similarity to C. jejuni and C. coli sequences and 95 to 98% similarity to sequences of other thermophilic Campylobacter species, such as C. lari and C. upsaliensis. The presence of C. coli DNA was detected by specific PCR in the samples from chicks obtained from a commercial hatchery; however, no Campylobacter was detected by culturing. In order to determine whether the same strains of bacteria were present in multiple levels of the integrator, we cultured Campylobacter sp. from a flock of broiler breeders and their 6-week-old progeny that resided on a commercial broiler farm. The broiler breeders had been given fluoroquinolone antibiotics, and we sought to determine whether the same fluoroquinolone-resistant strain was present in their progeny. The isolates were typed by pulsed-field gel electrophoresis, which confirmed that the parental and progeny flocks contained the same strain of fluoroquinolone-resistant C. coli. These data indicate that resistant C. coli can be present in multiple levels of an integrated poultry system and demonstrated that molecular techniques or more sensitive culture methods may be necessary to detect early colonization by Campylobacter in broiler chicks.     

Application of MALDI-TOF mass spectrometry for differentiation of closely related species of the “Arthrobacter crystallopoietes” phylogenetic group

MALDI mass spectra were generated for the type strain of Arthrobacter crystallopoietes VKM Ac-1107^T and for closely related (99.6?C100% 16S rRNA gene similarity) halotolerant Arthrobacter strains, as well as for some other Arthrobacter species. Results of the cluster analysis of the spectra were in agreement with the genotypic characteristics of bacteria (DNA-DNA hybridization and BOX-PCR). The data obtained in this study indicate that the halotolerant strains belong to two new Arthrobacter species. Specific peaks which can serve as chemotaxonomic markers of the species composing the phylogenetic group ??Arthrobacter crystallopoietes?? were revealed.     

Halomonas qiaohouensis sp. nov., isolated from salt mine soil in southwest China

Two Gram-stain negative, moderately halophilic, aerobic, motile bacteria, designated strains YIM QH88^T and YIM QH103, were isolated from the Qiaohou salt mine in Yunnan, southwest China. Cells of the strains were observed to be rod-shaped and produce creamy-coloured colonies. Growth of the two strains was observed at 10–45 °C (optimum 25–37 °C), at pH 6.0–10.0 (optimum 7.0–8.0), and in the presence of 0.5–20 % (w/v) NaCl (optimum 2–6 %). The two strains were found to contain summed feature 8 (C18:1 ω7c/ω6c), C19:0 cyclo ω8c and C16:0 as the major cellular fatty acids. The polar lipids were identified as diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and unknown phospholipid. The G+C content of the genomic DNA of strains YIM QH88^T and YIM QH103 were determined to be 64.6 and 64.2 mol%, respectively, and the predominant respiratory quinone detected was ubiquinone 9. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strains YIM QH88^T and YIM QH103 formed a distinct lineage within the genus Halomonas and were most closely related to Halomonas pantelleriensis DSM 9661^T with 97.3 and 97.5 % of 16S rRNA sequence similarity respectively. The DNA–DNA hybridization relatedness value for strains YIM QH88^T and YIM QH103 was 95.2 ± 0.8 %. The levels of DNA–DNA relatedness between each of these two strains and the type strains of phylogenetically closely related Halomonas species were clearly below 70 %. On the basis of their phylogenetic analysis, DNA–DNA hybridization relatedness, phenotypic and chemotaxonomic characteristics, strains YIM QH88^T and YIM QH103 should be classified as a novel species of the genus Halomonas, for which the name Halomonas qiaohouensis sp. nov. is proposed. The type strain is YIM QH88^T (=DSM 26770^T =CCTCC AB 2012965^T).