Molecular phylogeny and taxonomy of colorless, filamentous sulfur bacteria of the genus Thiothrix

Comprehensive investigation combining molecular genetic techniques and comparative studies of morphological and physiological properties made it possible to resolve the disputed issue of the taxonomic status of the groups ??T. nivea?? and ??Eikelboom type 021N?? of the genus Thiothrix. The phylogenetic trees constructed on the basis of 16S rRNA and gyrB gene sequences demonstrated that members of the genus Thiothrix formed a cluster within the order Thiotrichales. According to the ??ribosomal?? tree, the cluster of the genus Thiothrix was divided into two main groups, I and II, corresponding to the groups ??T. nivea?? and ??Eikelboom type 021N??. The levels of similarity between the 16S rRNA gene sequences of Thiothrix species reached 88.9?C100%. On the contrary, in the ??gyrase?? tree, these species were not divided into ??T. nivea?? and ??Eikelboom type 021N?? groups. The levels of similarity between the amino acid sequences of the gyrB gene fragments of Thiothrix species varied from 74.5 to 99.2%. Importantly, members of the groups ??T. nivea?? and ??Eikelboom type 021N?? formed very similar 16S rRNA secondary structures in the variable region V3, where a 30-nucleotide deletion characteristic of all Thiothrix species was detected. Phenotypic analysis of the studied bacteria revealed some morphological and physiological properties shared by the groups ??T. nivea?? and ??Eikelboom type 021N??. The data obtained indicate that members of the groups ??T. nivea?? and ??Eikelboom type 021N?? are phenotypically and genetically heterogeneous species within the single monophyletic genus Thiothrix..     

Quantification of an Eikelboom type 021N bulking event with fluorescence in situ hybridization and real-time PCR

Primers targeting 16S rRNA genes were designed to detect and quantify Eikelboom type 021N organisms by real-time PCR. Eikelboom type 021N filamentous bulking was induced in a laboratory-scale sequencing batch reactor and the evolution of Eikelboom type 021N 16S rRNA and 16S rRNA genes was monitored. A significant correlation was found between the sludge volume index and the amount of these filamentous organisms present in the sludge (r ²=94.6%, n=10, P<0.01), as measured by real-time PCR. The amount of Eikelboom type 021N 16S rRNA genes increased by a factor of 21 during the experiment, while the 16S rRNA increased by a factor of 33. Moreover, Eikelboom type 021N 16S rRNA increased with increased feeding frequency. It was observed that the RNA:DNA ratio peaked before the sludge volume index increased. In parallel, a fluorescence in situ hybridization study indicated a factor of four increase in the length of Eikelboom type 021N filaments, due to a factor of two increase in both length and number of Eikelboom type 021N filaments. Further, an increase in the fraction of filaments extending outside the activated sludge flocs was observed (19–55%). Monitoring of 16S rRNA genes and 16S rRNA of Eikelboom type 021N was shown to be valuable in evaluating activated sludge settling characteristics; and measuring RNA:DNA ratios may be used as an early warning tool for sludge bulking.     

Isolation and characterization of filamentous bacteria present in bulking activated sludge

Summary Several types of filamentous microorganisms were observed and identified in samples of poorly settling (bulking) activated sludge. The major types encountered and the frequency (percentage) of appearance in the total of all treatment plants sampled were: Eikelboom type 0041 (60), type 1701 (45), Haliscomenobacter hydrossis (35), type 021N (30), Thiothrix spp. (20), and Sphaerotilus natans (20). Isolation techniques and culture media were developed and used to recover 42 axenic strains of filamentous bacteria from sludge samples collected. The isolates were identified as strains of Thiothrix, Beggiatoa, S. natans, and Eikelboom types 021N, 1701, 0041, and 0803. Nutritional and differential characterization of the bacteria was important to the differentiation of groups which could not be easily distinguished on the basis of morphology. Although certain treatment plant operating parameters (organic loading) seemed associated with the presence of specific filamentous organism types, possible interaction among factors precluded definite establishment of a cause and effect relationship for most of the treatment plant characteristics and organisms observed.     

Overexpression in Escherichia coli of the AT-rich trpA and trpB genes from the hyperthermophilic archaeon Pyrococcus furiosus

Micromanipulation was used to obtain an isolate (BEN 52) of Eikelboom Type 1851 from a bulking activated sludge plant. Its 16S rDNA sequence reveals its closest relative is 'Roseiflexus castenholzii', a member of the phylum 'Chloroflexi', class 'Chloroflexi', previously called the green non-sulfur bacteria. The 16S rRNA targeted oligonucleotide probe designed for fluorescence in situ hybridisation against this sequence successfully identified filamentous bacteria with the morphological features of Type 1851 in activated sludge samples from plants in several countries and different operational configurations.     

The filamentous morphotype Eikelboom Type 1863 is not a single genetic entity

Five isolates of a filamentous bacterial morphotype with the distinctive diagnostic microscopic features of Eikelboom Type 1863 were obtained from activated sludge sewage treatment plants in Victoria, Australia. On the basis of phenotypic evidence and 16S rDNA sequence data, these isolates proved to be polyphyletic. Two (Ben 06 and Ben 06C) are from the Chryseobacterium subgroup which is in the Cytophaga group, subdivision I of the Flexibacter – Cytophaga – Bacteroides phylum. Two (Ben 56 and Ben 59) belong to the genus Acinetobacter , and one (Ben 58) is a Moraxella sp., closest to Mor. osloensis . The significance of these findings to the reliance on microscopic features for identification of these filamentous bacteria in activated sludge is discussed.     

沙坡头地区根瘤菌DNA同源性及16SrDNA全序列

数值分类和多位点酶电泳分析表明,分离自宁夏沙坡头 地区的12株根瘤菌构成一个独立的表观群。对这一菌群进行了DNA同源性和群内中心菌株1 6SrDNA全序列分析。12个菌株的G+C mol%在56.4～62.2范围内;群内DNA同源性为72.3% ～9.5%,大于70%,属种内水平;中心株N220的16SrDNA全序列与参比菌株的序列比较,从 模拟系统发育树看出,它与三株土壤杆菌、三株根瘤菌的16SrDNA序列同源性在94.8%～99 .2%的相似性水平上构成一个分支,看来沙坡头地区这群根瘤菌是一个独立的新种群。     

Identification of Thiobacillus ferrooxidans strains based on restriction fragment length polymorphism analysis of 16S rDNA.

The 16S rDNA sequences from ten strains of Thiobacillus ferrooxidans were amplified by PCR. The products were compared by performing restriction fragment length polymorphism (RFLP) analysis with restriction endonucleases Alu I, Hap II, Hha I, and Hae III. The RFLP patterns revealed that T. ferrooxidans could be distinguished from other iron- or sulphur-oxidizing bacteria such as T. thiooxidans NB1-3, T. caldus GO-1, Leptospirillum ferrooxidans and the marine iron-oxidizing bacterium strain KU2-11. The RFLP patterns obtained with Alu I, Hap II, and Hae III were the same for nine strains of T. ferrooxidans except for strain ATCC 13661. The RFLP patterns for strains NASF-1 and ATCC 13661 with Hha I were distinct from those for other T. ferrooxidans strains. The 16S rDNA sequence of T. ferrooxidans NASF-1 possessed an additional restriction site for Hha I. These results show that iron-oxidizing bacteria isolated from natural environments were rapidly identified as T. ferrooxidans by the method combining RFLP analysis with physiological analysis.     

Filamentous Alphaproteobacteria associated with bulking in industrial wastewater treatment plants

The phylogeny and distribution of filamentous Alphaproteobacteria, morphologically similar to “Nostocoida limicola” and Eikelboom Type 021N that cause the solids separation problem of bulking in industrial activated sludge plants is described here. A combination of culture-dependent and culture-independent molecular methods has characterized 5 novel species. 16S rRNA targeted oligonucleotide probes were designed for their in situ identification by fluorescence in situ hybridisation (FISH) and used to monitor their presence in 86 WWTPs treating different industrial effluents in four European countries. The involvement of these bacteria in bulking in these plants was confirmed. Filaments hybridising with the ALF-968 probe for the Alphaproteobacteria were present in 65% of the WWTPs examined. They were dominant and therefore probably responsible for bulking in 25.5% of them. The heterogeneous filamentous alphaproteobacterial populations in these communities could be completely identified after application of the oligonucleotide probes used in this study in 91% of the plants containing them. The only filamentous Alphaproteobacteria retrieved in pure culture was isolated from three different industrial WWTPs plants. None of these isolates could grow anaerobically on glucose or denitrify, but all grew aerobically and heterotrophically on a range of carbon sources. Although morphologically similar to the Eikelboom Type 021N morphotype, they were not involved in sulphur metabolism. These bacteria accumulated lipidic storage granules that were associated with their presence under the unbalanced growth conditions existing in these plants.     

Organization of the ribosomal operon 165-235 gene spacer region in representatives of Neisseria gonorrhoeae

Ribosomal rRNA gene fragments (rDNA) encompassing part of the 16S rDNA, the 16S-23S rDNA spacer region and part of the 23S rDNA of 229 Neisseria gonorrhoeae strains were enzymatically amplified using conserved primers. The fragments of approximately 1200 bp were subjected to restriction analysis with HinfI. This revealed 13 patterns (patterns I-XIII) of which patterns I (78 strains), II (32 strains), III (38 strains) and IV (56 strains) were the most abundant, comprising 89.1% of the strains. The obtained restriction patterns consisted of 3 to 8 bands, ranging in size from 32 to 854 bp. The sum of the obtained bands was about 1200 bp for patterns I, II, III, IV, V, IX, and XIII. However, for patterns VI, VII, VIII, X, XI and XII, the sum of the bands well exceeded the estimated size of approximately 1200 bp. We demonstrated that this results from sequence divergence in the 4 rRNA operons, present in the genome of N. gonorrhoeae, giving rise to patterns that are a combination of several other patterns.     

甲烷氧化菌Methylomonas sp.GYJ3中甲烷单加氧酶基因与16S rRNA序列分析

甲烷氧化细菌中的关键酶系甲烷单加氧酶是一个含双核铁的多组份氧化酶,常温、常压下能够催化甲烷转化为甲醇。对甲烷氧化细菌Methylomonas sp.GYJ3中溶解性甲烷单加氧酶基因和16SrDNA进行了测序与分析。利用已知相关基因数据库信息,设计了PCR引物和测序引物,获得了满意的测序结果。全长的溶解性甲烷单加氧酶基因为5690bp,部分16S rDNA的序列长度为1280bp。与已发表的甲烷氧化细菌中甲烷单加氧酶进行了比较,结果表明MMOX组份中氨基酸序列的同一性为78%到99%,基因序列的同一性为71%到97%,6个组份中orfY片段的同一性相对较低。MMOX氨基酸序列的多序列联配表明,MMOX序列具有高度保守性,特别是在双核铁中心区域。16S rDNA进化分析显示Methylomonas sp.GYJ3与γ蛋白细菌是相关联的,基于MMOX氨基酸序列的进化分析证明,与Methylomonas sp.GYJ3最近似的菌株是Ⅰ型甲烷氧化细菌Methylomonas sp.KSWⅢ。综合分析表明,菌株GYJ3属于Ⅰ型甲烷氧化细菌Methylomonas sp.属。这个结果为Ⅰ型甲烷氧化细菌也能表达溶解性甲烷单加氧酶提供了新的证据。羟基化酶的理论等电点是6.28,理论分子量为248874.41Da。     

2株创伤弧菌的16S-23S rDNA间区的克隆、测序及分析

根据细菌的16SrDNA3’端和23SrDNA5’端的高度保守区设计引物,PCR扩增了2株创伤弧菌（Vibrio vulnificus）的16S-23SrDNA间区（Intergenic spacer,IGS）,克隆到pGEM-T载体上,测序。用BLAST和DNA star软件对16S-23SrDNA间区序列及其内的tRNA基因进行比较分析。结果表明,2株创伤弧菌共测出9条16S-23SrDNA间区序列,其中ZSU006测出5条,间区类型分别为：IGS^GLAV、IGS^GLV、IGS^LA、IGS^A和IGS^G.其中IGS^GLAv最大,包含tRNA^Glu、tRNA^Lys、tRNA^Ala。和tRNA^Val基因;IGS^GLV包含tRNA^Glu、tRNA^Lys。和tRNA^Val基因;IGS^LA,则包含tRNA^Ile和tRNA^Ala基因;IGS^G包含tRNA^Glu基因;而IGS^A仅包含tRNA^Ala基因。菌株CG021测出的16S-23SrDNA IGS序列有4条,除缺少IGS^A外,其余的IGS类型均与ZSU006的相同。与GenBank内的创伤弧菌ATCC27562的IGS序列比较,发现创伤弧菌所有类型的IGS的tRNA基因两端的非编码区具有较高的种内同源性。16S-23SrDNA间区结构的差异为建立一种新的创伤弧菌检测方法奠定了基础。     

Phylogeny of green sulfur bacteria on the basis of gene sequences of 16S rRNA and of the Fenna-Matthews-Olson protein

The phylogeny of green sulfur bacteria was studied on the basis of gene sequences of the 16S rRNA and of the Fenna-Matthews-Olson (FMO) protein. Representative and type strains (31 strains total) of most of the known species were analyzed. On the basis of fmoA gene sequences from Chlorobium tepidum ATCC 49652(T) and Chlorobium limicola DSM 249(T) available from the EMBL database, primers were constructed that allowed sequence analysis of a major part of the fmoAgene. The largely congruent phylogenetic relationship of sequences of the fmoA gene and of 16S rDNA gives considerable support to the phylogeny of green sulfur bacteria previously suggested on the basis of 16S rDNA sequences. Distinct groups of strains were recognized on the basis of 16S rDNA and FMO sequences and supported by characteristic signature amino acids of FMO. Marine strains formed clusters separate from freshwater strains. The resulting phylogenetic grouping and relationship of the green sulfur bacteria do not correlate with their current taxonomic classification.     

Identification of strains assigned to the genus Gluconobacter Asai 1935 based on the sequence and the restriction analyses of the 16S-23S rDNA internal transcribed spacer regions

Thirteen reference strains, including the type strains of the type species of the genus Gluconobacter, Gluconobacter oxydans (NBRC 14819T), Gluconobacter cerinus (NBRC 3267T), and Gluconobacter frateurii (IFO 3264T) were examined for their species identification based on the sequence and the restriction analyses of the 16S-23S rDNA internal transcribed spacer (ITS) regions. A phylogenetic tree constructed by the neighbor-joining method represented three clusters corresponding respectively to the three species, G. oxydans, G. cerinus, and G. frateurii. The type strain of Gluconobacter asaii (NBRC 3276T), which is a junior subjective synonym of G. cerinus, was included completely in the G. cerinus cluster. Several restriction endonucleases discriminating the three species from one another were selected by computer analyses: Bsp1286I, MboII, SapI, Bpu10I, EarI, BsiHKAI, and FatI. On digestion of the PCR products with restriction endonucleases Bsp1286I and MboII, all the restriction patterns coincided with those of the type strains of the three species except for strain NBRC 3251. This strain gave a different pattern from the type strain of G. frateurii, when digested with MboII. However, strain 3251 was included phylogenetically in the G. frateurii cluster. All the reference strains were thus identified at the species level by the sequence and the restriction analyses of the 16S-23S rDNA ITS regions.     

新疆地区盐湖的中度嗜盐菌16S rDNA全序列及DNA同源性分析

通过数值分类和16S rDNA PCR-RFLP分析,对分离自新疆地区的中度嗜盐革兰氏阴性菌进行研究,发现了一个新类群。在此基础上,进行了中心株AI－3的16S rDNA全序列分析,并与中度嗜盐菌已知种和相关种进行比较,得到系统发育树状图。在此树状图中,大多数参比菌株聚在一起,其16S rDNA全序列的同源性在96％以上,而AI－3与参比菌株的16S rDNA全序列相比,其相似性低于75％。但是,AI－3与Alcanivorax borkumensis^[1]的16S rDNA全序列的相似性为96％,与Halobacillus litoralis的16S rDNA全序列的相似性为99％,三者构成一个独立的发育分支。这说明在系统发育上,AI－3与参比菌株属于不同的分支,是一个新的类群。在新类群内,菌株之间的DNA同源性大于70％,而中心株AI－3与标准菌株伸长盐单胞菌（Halomonas elongata)的DNA同源性为44％,表明新分离的菌株可能构成一个新种群。     

Quantification of Hyphomicrobium populations in activated sludge from an industrial wastewater treatment system as determined by 16S rRNA analysis

The bacterial community structure of the activated sludge from a 25 million-gal-per-day industrial wastewater treatment plant was investigated using rRNA analysis. 16S ribosomal DNA (rDNA) libraries were created from three sludge samples taken on different dates. Partial rRNA gene sequences were obtained for 46 rDNA clones, and nearly complete 16S rRNA sequences were obtained for 18 clones. Seventeen of these clones were members of the beta subdivision, and their sequences showed high homology to sequences of known bacterial species as well as published 16S rDNA sequences from other activated sludge sources. Sixteen clones belonged to the alpha subdivision, 7 of which showed similarity to Hyphomicrobium species. This cluster was chosen for further studies due to earlier work on Hyphomicrobium sp. strain M3 isolated from this treatment plant. A nearly full-length 16S rDNA sequence was obtained from Hyphomicrobium sp. strain M3. Phylogenetic analysis revealed that Hyphomicrobium sp. strain M3 was 99% similar to Hyphomicrobium denitrificans DSM 1869(T) in Hyphomicrobium cluster II. Three of the cloned sequences from the activated sludge samples also grouped with those of Hyphomicrobium cluster II, with a 96% sequence similarity to that of Hyphomicrobium sp. strain M3. The other four cloned sequences from the activated sludge sample were more closely related to those of the Hyphomicrobium cluster I organisms (95 to 97% similarity). Whole-cell fluorescence hybridization of microorganisms in the activated sludge with genus-specific Hyphomicrobium probe S-G-Hypho-1241-a-A-19 enhanced the visualization of Hyphomicrobium and revealed that Hyphomicrobium appears to be abundant both on the outside of flocs and within the floc structure. Dot blot hybridization of activated sludge samples from 1995 with probes designed for Hyphomicrobium cluster I and Hyphomicrobium cluster II indicated that Hyphomicrobium cluster II-positive 16S rRNA dominated over Hyphomicrobium cluster I-positive 16S rRNA by 3- to 12-fold. Hyphomicrobium 16S rRNA comprised approximately 5% of the 16S rRNA in the activated sludge.     

油菜内生细菌16S核糖体DNA的RFLP分析

植物内生细菌定殖在植物组织内部但不引起明显的病害症状。从健康油菜植株的不同器官中分离到大量内生细菌,这些细菌菌落形态存在明显的差异,表明油菜组织中存在大量内生细菌,且类群丰富。分离到的122株内生细菌根据菌落形态可以划分为35类;利用细菌通用引物对16S核糖体DNA进行扩增, 获得约15 kb片段,分别用内切酶HaeⅢ和MspⅠ对扩增产物进行限制性酶切,产生不同的酶切图谱,根据酶切图谱聚类分析结果,所有供试菌株被归为39类,这一结果从遗传上显示油菜内生细菌类群的多样性。两种方法归类结果比较发现菌落特征所反映的信息量有限,只能作为初步的参考指标,核糖体DNA的限制性片段长度多态性分析快速、准确,可以作为油菜内生细菌多样性分析的一种有效方法     

分离自补骨脂等宿主根瘤的24株菌的数值分类和16S rDNA PCR-RFLP 研究

采用数值分类和16S rDNA PCR-RFLP对分离自云南省豆科植物补骨脂(Psoralea corylifolia)、葛藤(Pueraria lobata)、杭子梢(Campylotropis macrocarpa)等宿主的24株菌及10株根瘤菌参比菌株进行了研究。数值分类结果表明, 在84%相似性水平上, 所有的菌株可分为3群：群Ⅲ为未知菌群, 群Ⅰ为慢生菌群, 群Ⅱ为快生和中慢生菌群。从依据16S rDNA PCR-RFLP分析建立的树状图来看, 在70%相似性水平上, 所有的菌株可分为5个系统发育分支：分支Ⅰ和Ⅴ没有参比菌株, 为未知分支; 分支Ⅱ为Agrobacterium-Sinorhizobium-Rhizobium, 分支Ⅲ为Mesorhizo- bium, 分支Ⅳ为Bradyrhizobium。数值分类和16S rDNA PCR-RFLP的结果部分一致, 有2株菌与A. tumefaciens IAM13129T聚在一起。     

分离自补骨脂等宿主根瘤的24株菌的数值分类和16S rDNA PCR-RFLP 研究

采用数值分类和16S rDNA PCR-RFLP对分离自云南省豆科植物补骨脂(Psoralea corylifolia)、葛藤(Pueraria lobata)、杭子梢(Campylotropis macrocarpa)等宿主的24株菌及10株根瘤菌参比菌株进行了研究.数值分类结果表明,在84%相似性水平上,所有的菌株可分为3群:群Ⅲ为未知菌群,群Ⅰ为慢生菌群,群Ⅱ为快生和中慢生菌群.从依据16S rDNA PCR-RFLP分析建立的树状图来看,在70%相似性水平上,所有的菌株可分为5个系统发育分支:分支Ⅰ和Ⅴ没有参比菌株,为未知分支;分支Ⅱ为Agrobacterium-Sinorhizobium-Rhizobium,分支Ⅲ为Mesorhizobium,分支Ⅳ为Bradyrhizobium.数值分类和16S rDNA PCR-RFLP的结果部分一致,有2株茵与A.tumefaciens IAM13129T聚在一起.     

Analysis of genomic diversity among photosynthetic stem-nodulating rhizobial strains from northeast Argentina

The genomic diversity among photosynthetic rhizobia from northeast Argentina was assessed. Forty six isolates obtained from naturally occurring stem and root nodules of Aeschynomene rudis plants were analyzed by three molecular typing methods with different levels of taxonomic resolution: repetitive sequence-based PCR (rep-PCR) genomic fingerprinting with BOX and REP primers, amplified 16S rDNA restriction analysis (ARDRA), and 16S-23S rDNA intergenic spacer-restriction fragment length polymorphism (IGS-RFLP) analysis. The in vivo absorption spectra of membranes of strains were similar in the near infrared region with peaks at 870 and 800 nm revealing the presence of light harvesting complex I, bacteriochlorophyll-binding polypeptides (LHI-Bchl complex). After extraction with acetone-methanol the spectra differed in the visible part displaying peaks belonging to canthaxanthin or spirilloxanthin as the main carotenoid complement. The genotypic characterization by rep-PCR revealed a high level of genomic diversity among the isolates and almost all the photosynthetic ones have identical ARDRA patterns and fell into one cluster different from Bradyrhizobium japonicum and Bradyrhizobium elkanii. In the combined analysis of ARDRA and rep-PCR fingerprints, 7 clusters were found including most of the isolates. Five of those contained only photosynthetic isolates; all canthaxanthin-containing strains grouped in one cluster, most of the other photosynthetic isolates were grouped in a second large cluster, while the remaining three clusters contained a few strains. The other two clusters comprising reference strains of B. japonicum and B. elkanii, respectively. The IGS-RFLP analysis produced similar clustering for almost all the strains. The 16S rRNA gene sequence of one representative isolate was determined and the DNA sequence analysis confirmed the position of photosynthetic rhizobia in a distinct phylogenetic group within the Bradyrhizobium rDNA cluster.     

Molecular characterisation of Halobacillus strains isolated from different medieval wall paintings and building materials in Austria

We previously reported on the detection and isolation of an indigenous population of Halobacillus from salt-damaged medieval wall paintings and building materials of Herberstein castle in St. Johann bei Herberstein in Styria, Austria. Several moderately halophilic, Gram-positive, endospore-forming Halobacillus-like bacteria could be again isolated by conventional enrichment from salt efflorescences collected in the medieval St. Virgil's chapel in Vienna. Comparative 16S rDNA sequence analyses showed that the St. Virgil isolates are most closely related (>98.5% sequence similarity) to Halobacillus trueperi, Halobacillus litoralis, and to our previous halobacilli strains obtained from the castle Herberstein. Based on 16S rDNA sequence analysis, the strains could be clustered in three different groups. Group I: St. Virgil strains S3, S4, S21, and S22 (99.8–100% sequence similarity); group II: Herberstein strains K3-1, I7, and the St. Virgil strain S20 (99.3–99.7% sequence similarity); and group III: Herberstein strains I3, I3A, and I3R (100% sequence similarity). Molecular typing by denaturing gradient gel electrophoresis (DGGE), random amplified polymorphic DNA (RAPD-PCR), and internal transcribed spacer-homoduplex–heteroduplex polymorphism (ITS-HHP) fingerprinting showed that all isolates are typeable by each of the methods. RAPD was the most discriminatory method. With respect to their physiological characteristics—i.e., growth in the presence of 5–20% (w/v) NaCl, no growth in the absence of NaCl, optimum growth at 37 °C in media containing 5–10% (w/v) NaCl, and optimum pH around 7.5–8.0—the St. Virgil isolates resembled our previously isolated strains. However, the St. Virgil strains showed some differences in their biochemical properties. St. Virgil isolates hydrolysed Tween 80, two isolates reduced nitrate, and no isolate liquefied gelatine. The recurrent isolation of halobacilli from salt efflorescences on historic buildings and monuments at two different geographical locations may indicate that this group of bacteria is common in salt-affected ruins.