International Journal of Systematic Bacteriology (1998), 48, 11 29-1 143 Printed in Great Britain

Phylogenetic relationships among the , their taxonomic reclassification and description of the new genera , , Isochromatium, Marichromatiurn , Thiococcus, Thiohalocapsa and

Johannes F. Imhoff, Jorg Wing and Ralf Petri

Author for correspondence: Johannes F. Imhoff. Tel: +49 431 697 3850. Fax: +49 431 565876. e-mail : jimhoff(n ifm.uni-kiel.de

lnstitut fur Meereskunde Sequences of the 16s rDNA from all available type strains of an der Universitat Kiel, have been determined and were compared to those of other Abteilung Marine Mikrobiologie, Chromatiaceae, a few selected and Escherichia coli. Dusternbrooker Weg 20, The clear separation of Ectothiorhodospiraceae and Chromatiaceae is D-24105 Kiel, Germany confirmed. Most significantly the sequence comparison revealed a genetic divergence between Chromatium species originated from freshwater sources and those of truly marine and halophilic nature. Major phylogenetic branches of the Chromatiaceae contain (i)marine and halophilic species, (ii)freshwater Chromatium species together with Thiocystis species and (iii)species of the genera Thiocapsa and Amoebobacter as recently reclassified [Guyoneaud, R. & 6 other authors (1998). lnt J Syst Bacteriol48, 957-9641, namely Thiocapsa roseopersicina, Thiocapsa pendens (formerly Amoebobacter pendens), Thiocapsa rosea (formerly Amoebobacter roseus), Amoebobacter purpureus and Thiolamprovum pedioforme (formerly Amoebobacter pedioformis). The genetic relationships between the species and groups are not in congruence with the current classification of the Chromatiaceae and a reclassification is proposed on the basis of 16s rDNA sequence similarity supported by selected phenotypic properties. The proposed changes include the transfers of Chromatium minus and Chromatium violascens to Thiocystis minor comb. nov. and Thiocystis violascens com b. nov., of Chromatium vinosum, Chromatium minutissimum and Chromatium warmingii to the new Allochromatium as Allochromatium vinosum comb. nov., Allochromatium minutissimum comb. nov., and Allochromatium warmingii comb. nov., of Chromatium tepidum to the new genus Thermochromatium as Thermochromatium tepidum comb. nov., of Chromatium salexigens and Chromatium glycolicum to the new genus Halochromatium as Halochromatium salexigens comb. nov. and Halochromatium glycolicum comb. nov., of Chromatium gracile and Chromatium purpuratum to the new genus as Marichromatium gracile comb. nov. and Marichromatium purpuratum comb. nov., of Thiocapsa pfennigii to Thiococcus pfennigii gen. nom. rev., of Thiocapsa halophila to the new genus Thiohalocapsa as Thiohalocapsa halophila comb. nov., and of Chromatium buderi to the new genus lsochromatium as lsochromatium buderi comb. nov.

~ Keywords : phylogeny, Chromatiaceae, taxonomic reclassification

The EMBL accession numbers for the 16s rDNA sequences determined in this study are given in Table 1. Downloaded from www.microbiologyresearch.org by IP: 134.245.215.185 1129 On: Tue, 30 May 2017 10:29:44 J. F. Imhoff, J. Suling and R. Petri

INTRODUCTION Strains were cultivated in synthetic medium according to Pfennig & Triiper (1992). The final pH was 7.2. For marine Historically, the of phototrophic purple and halophilic strains, 1-10% NaCl was added to the sulfur , has been based on easily recognizable medium, as required. Pure cultures were grown in 100 ml phenotypic properties (see Pfennig & Triiper, 1974). screw-capped bottles completely filled with synthetic me- The traditional taxonomic system of these bacteria dium and incubated in the light. is not phylogenetically oriented. It is primarily of PCR amplification and 16s rDNA sequencing and analysis. practical relevance, and inconsistencies between the Cell material for 16s rDNA sequencing was taken from taxonomic treatment and genetic relationships of the 1-2 ml of well-grown liquid cultures. DNA was extracted have been discussed previously and purified by using the QIAGEN genomic DNA buffer (Gibson et al., 1979; Fowler et al., 1984; Stackebrandt set. Recombinant Taq polymerase was used for PCR (Mullis et al., 1984; Truper, 1987). Indeed, phylogenetic & Faloona, 1987) which was started with the following information became available only with the estab- primers : 5’-GTTTGATCCTGGCTCAG-3’ and 5’-TACC- TTGTTACGACTTCA-3’ (positions 1 1-27 and 1489-1 506, lishment of sequencing techniques for proteins and respectively, according to the Escherichia coli 16s rRNA nucleic acids on the basis of sequences of cytochrome numbering of the International Union of Biochemistry). The c (Ambler et al., 1979; Dickerson, 1980) and oligo- PCR products were purified by using the QIAquick PCR nucleotides obtained from 16s rRNA digestion with purification kit. Sequences were obtained by cycle specific endonucleases (Gibson et al., 1979; Fowler et sequencing with the SequiTherm sequencing kit (Biozym) al., 1984; Stackebrandt et al., 1984), later by sequences and the chain-termination reaction (Sanger et a/., 1977) of the 16s rRNA gene. It was obvious already from the using an automated laser fluorescence sequencer early investigations on the genetic relationships that (Pharmacia). Sequences were aligned using the CLUSTAL w phototrophic bacteria are found in several major program (Thompson et a/., 1994). The alignment length was branches of a sequence-based phylogenetic bacterial from positions 29 to 1381 according to the Escherichia coli numbering (including gaps 1400 positions). The distance system (see Woese, 1987), in part separated from and matrix was calculated on the basis of the algorithm of Jukes in part intermixed with non-phototrophic bacteria. & Cantor (1969) with the DNADIST program within the Among the phototrophic , separate PHYLIP package (Felsenstein, 1989). The FITCH program in phylogenetic lines are represented by the purple sulfur the PHYLIP package fitted a tree to the evolutionary distances. bacteria (Chromatiaceae and Ectothiorhodospiraceae) Nucleotide sequence accession numbers. The 16s rDNA in the y- and by the purple non-sulfur sequences determined in this study have been deposited in bacteria in the a-Proteobacteria and the P-Proteo- the EMBL database under accession numbers given in Table bacteria. First surveys on the relationships of purple 1. Accession numbers of additional 16s rDNA sequences of sulfur bacteria have been made by comparison of Chromatiaceae that were published previously are also oligonucleotide catalogues (Fowler et al., 1984; contained in Table 1. Further sequences (accession nos in Stackebrandt et al., 1984). More recently, sequence parentheses) from Ectothiorhodospiraceae and Escherichia analyses and comparisons lead to the separation of the coli (K02555) were available from the EMBL database: Ectothiorhodospiraceae into two genera that can also Ectothiorhodospira mobilis strain DSM 237T (X9348 1), be recognized by their salt responses and other Ectothiorhodospira haloalkaliphila strain ATCC 5 1935’ (X93479), Ectothiorhodospira shaposhnikovii strain DSM phenotypic properties (Imhoff & Suling, 1996). 16s 239 (X93480), halophila strain DSM 244’ rDNA sequences of only a few species of the (M26630), and Halorhodospira halochloris DSM 1059T Chromatiaceae were available until recently (DeWeerd (M59152) (see Imhoff & Suling, 1996). et al., 1990; Wahlund et al., 1991; Caumette et al., 1997; Guyoneaud et al., 1997, 1998). In the present RESULTS work 16s rDNA sequences of all available type strains of Chromatium species are compared to related purple The relationships of the investigated type strains of sulfur bacteria and used to characterize their genetic Chromatium species and related purple sulfur bacteria relationships. On this basis and with the additional based on almost complete sequences of the 16s rDNA support of phenotypic properties a reclassification of is shown in a distance matrix with similarity values and these bacteria is proposed. A positive correlation Knucvalues (Table 2) and a phylogenetic tree, based on between differences in several phenotypic properties to the data from this distance matrix (Fig. 1). similarities of 16s rDNA sequences is discussed and in It is quite apparent from their genetic relationships particular these properties are considered to be of that a major deeply branching cluster with marine and importance for the classification of these bacteria. halophilic Chromatiaceae species is clearly separated from another branch with predominantly freshwater METHODS species. Within this latter branch, one cluster contains Thiocapsa and Amoebobacter species according to Source and culture of bacterial strains. All strains of purple Guyoneaud et al. (1998), while another one includes sulfur bacteria used for this study are listed in Table 1, which freshwater Chromatiurn species together with known in addition shows the previously used and the newly Thiocystis species. proposed names, the original strain designations, the DSM numbers when available and the EMBL accession numbers The first branch consists of Chromatium species which for their 16s rDNA sequences. Cultures of all strains are have been isolated from marine and hypersaline maintained in our laboratory (see Table 1). environments and which specifically and character-

~~~~ ______~~ Downloaded from www.microbiologyresearch.org by 1130 IP: 134.245.215.185 International Journal of Systematic Bacteriology 48 On: Tue, 30 May 2017 10:29:44 Phylogenetic taxonomy of Chrornatiaceae

Table 1. Species and strains of Chromatiaceae compared in this study , . . , ., . , ...... , ...... , . , ...... , ., ......

Type strains are denoted by T; NN, not available in this collection.

Previous name New name DSM Other Authors’ EMBL no. no. designation(s) no.

~~ ~~ Chrotnutiurn okenii Unchanged 169T 1111 6010 Y 12376 Chrorncitiurn vinosum Allochromatiuni vinosum 1SOT D 51 10 M26629 Cli r omci t iunz nz inu t issim um Allochromatiuni ininutissiinum 1376’ MSU 5310 Y 12369 Chroniutiurn warmingii Allochromatium warmingii 173T 6512 5810 Y 12365 Chr onwt i urn t epidum Thermochromatiuni tepidum 3771T MC NN M59 150 Chr oni~t i uin in in us Thiocystis minor 17ST 121 1 5710 Y 12372 Chr oina t I um violascens Thiocys t is violascens 1 6ST 6111 5410 A5224438 Chromit I uin purpura t urn Marichromatiuni purpuratum 1591T 984 5500 A5224439 Chrotnutiurn gracile Marichromatiuin gracile 203T 861 1 5210 x93473 Chrotnatiuin huderi Isochromutiuni huderi 176’ 8111 5610 A5224430 Chrotnutiurn salexigens Hulochrortiatiuni salexigens 4395* SG320 1 63 10 X98597 Clirotnut iuin glycolicum Halochromatiurn glycolicum 11080’ SL320 1 6410 X93472 Thiocqm I pfennig ii Tliiococcus p fennigii 226 801 3 4250 Y 12373 Thiocup,s 1 i halophila Thiohalocapsa halophila 62 1 OT SG3202 4270 A5002796 Thio rh or10 vibr io winogradskji Unchanged 6702T 0651 1 4910 Y 12368 Thiocj3.st I s violacea Unchanged 207’ 271 1 4340 Y 11315 Thiocjystis gelutinosa Unchanged 215’ 261 1 4310 Y11317 Rhabdoc‘liroma tium inarinurti Unchanged 526 1 8315 6510 X843 16 minus Thiorhodococcus minor” 1 151ST CE2203 6680 Y11316 Amoehohucter purpureus Unchanged 4197’ ThSchl2 4450 Y 12366 Tliio caps^ I roseope rs icin a Unchanged 217’ 171 1 4210 Y 12364 Amoehohucter pendens Thiocapsa pendens? 236’ 1314 4410 A5002797 * Corrected epithet. I- See Guyoneaud et al. (1998).

istically require various concentrations of salt for subcluster to which Chromatium tepidum is distantly growth . Chronm t ium salexigens and Chromatium related. A second subcluster is represented by glycolicwn form one line. Chromatium gracile and Chroma t ium ni in us, Thioc ys t is vioIacea , Thio c y st is Chromatium purpuratum form a second line together gela tinosa and Chromat ium violascens. Chromatium with an isolate that has been first described as okenii, the type species of the genus Chromatium, is ‘ Rhodohurter marinus’ (Burgess et al., 1994 ; 16s only distantly related to other bacteria of this cluster. rDNA sequence of this strain as personal communi- Chromatium weissei, which is phenotypically quite cation of T. Matsunaga). Also, Thiocapsapfennigii and similar to , also has a number of Thiocapstr halophila belong to this cluster and therefore significant positions in its 16s rDNA sequence in certainly can no longer be classified as Thiocapsa common specifically with Chromatium okenii, as could species (Guyoneaud et al., 1998). In addition, be delineated from the comparison of its oligo- Thiorhodovibrio winogradskyi, Rhabdochromatium nucleotide catalogue (Fowler et al., 1984) with the marinum. Thiorhodococcus minor and Chromatium sequence of Chromatium okenii. A pure culture of buderi form well-separated lines within this cluster Chromatiurn weissei is at present not available and (Fig. 1). therefore the comparison of its complete sequence was The second branch mostly contains species that orig- not possible. inate from freshwater habitats and is significantly The second cluster of this branch showed some distant distinct from the branch of truly marine and halophilic relationships to the freshwater Chromatium species. In species. On the basis of the sequence data, two major this cluster are Thiolamprovum pedioforme (formerly clusters can be recognized, one with Chromatium and Amoebobacter pedioformis), Amoehohacter purpureus, Thiocj*stis species, the other with Thiocapsa and Thiocapsa pendens (formerly Amoebobacter pendens), Amoehohacter species, including Thiolamprovum Thiocapsa rosea (formerly Amoebobacter roseus) and pediofornw (formerly Amoebobacter pedioformis). Thiocapsa roseopersicina (Fig. 1 ; Guyoneaud et al., In the first cluster Chromatiurn warmingii, Chromatium 1998). Although most of these species characteristi- vinosum and Chromatium minutissimum form one cally are found in freshwater habitats, a number of

~ -~ ~ Downloaded from www.microbiologyresearch.org- by lnterna tiona I Jo urnaI of Systematic Bacteriology 48 IP: 134.245.215.185 1131 On: Tue, 30 May 2017 10:29:44 27, salexigens DSM warmingii 9, DSM & The values on the are upper right the uncorrected sequence similarities 14, bacteria Table 6 5 4 3 2 0 9 8 6 4 3 2 7 5 1 1 Cantor (1969). 1, Organisms: Thiocapsa rosea Thiocapsa cohohdsia vacuolata Ectothiorhodospira .0 007 .0 009 .9 019 ,8 0.106 0,086 0,092 0.109 0.094 0.093 0.083 0.099 0.095 0.103 0.095 0.097 0.101 0.102 0.113 0.100 0.097 0.104 0.099 0.089 0.104 0.098 0.105 0.109 0.095 0.104 0.093 0.109 0-102 0.091 0.098 Thiocystis violacea .1 0.097 0.113 90.07 0.107 0.138 0.121 0.129 0.126 0.119 0.133 0.070 0-118 0.077 0.107 0.064 0.115 0.073 0.082 0.096 0.082 0.095 0.102 0.102 0.1 0.050 0.086 0.093 .0 001 51 9.3 10 9.8 89.10 93.08 91.08 90.03 91.03 92.23 95.19 90.43 90.69 91.89 91.41 0.091 0.104 0.062 1 237T; 3 6702T; I5 2. .9 006 .9 9.1 26 88.14 92.65 90.61 0.090 0.096 0.099 0,138 40 9.0 12 8.2 05 9.4 89.34 92.24 90.50 89.32 91.27 90.30 94.07 Levels and DSM DSM 2 23, 1, 0.112 0.122 0.119 aohdsia halochloris Halorhodospira Escherichia 3 Rhabdochromatium marinum Rhabdochromatium 4395T; 173T; 19, of DSM 0.102 0.106 0.126 0.123 165 4 DSM 207T; 15, 5, 235T; 10, 0,093 0.112 0.092 0.112 0.096 0.099 0.110 0,131 0.100 91.49 rDNA Isochromatium buderi Isochromatium Thermochromatium tepidum 5 coli .0 0.076 0.102 0.120 0.094 0.104 0.130 0.063 0.112 0.124 09 9.2 88.98 93.82 90.98 7 6 Marichromatium sequence as DSM Thiolamprovum 0.101 88.83 93.35 reference Chromatium 2 1 1 0,093 0.093 8 lT; DSM similarity DSM .0 014 .2 9.3 54 9.4 46 9.7 12 9.0 10 9.6 22 9.2 25 8.2 89 8.7 85 8.0 77 8.0 64 86.54 86.41 86.70 87.71 87.80 88.56 88.97 88.95 85.32 92.53 92.32 92.28 90.16 91.05 91.20 91.20 93.37 94.60 94.44 95.42 96.13 0,108 0.027 0.114 0.102 .0 015 .6 006 .7 002 .7 9.0 37 9.4 88 8.1 25 9.3 37 8.6 94 8.5 81 8.6 83 8.6 72 86.60 87.29 87.56 85.88 88.39 86.05 88.06 85.76 88.16 86.84 89.05 89.44 87.16 87.58 85.36 88.22 93.74 88.19 92.03 83.68 92.53 91.75 89.41 91.18 88.89 92.01 93.04 89.69 93.79 91.03 98.40 90.39 90.55 92.33 92.99 0.074 0.062 0.070 0.056 0.070 0.068 0.115 0.065 0.105 0.058 0.059 0.121 0.104 0,106 0,104 .0 018 73 9.2 47 9.9 34 9.3 18 9.8 01 8.9 18 9.6 20 8.1 82 8.3 79 8.8 69 8.5 61 86.86 86.13 86.15 86.99 83.90 88.08 83.82 87.90 84.76 88.03 85.47 88.28 84.98 84.11 85.55 92.04 85.97 91.76 83.16 91.82 90.94 89.69 90.68 90.17 89.69 91.08 86.34 91.87 87.69 88.46 92.93 87.52 93.49 94.29 88.50 94-77 89.31 95.42 88.79 97.38 90.12 88.58 89.40 89.10 0.118 0.106 0.102 39 9.3 12 9.9 91.08 90.89 91.26 91.13 93.99 96 9.0 05 9.1 05 9.1 18 9.7 18 8.7 85 9.5 02 9.2 42 9.8 56 8.4 91 8.1 78 8.3 69 8.7 86.95 86.97 86.98 85.72 87.73 85.65 87.85 86.16 88.41 85.30 85.30 89.12 85.21 85.96 89.34 84.80 86.56 85-67 86.31 85.25 86.20 93.38 86.53 86.42 85.58 94.24 86.24 86.38 84.15 94.52 87.93 86.03 90.18 88.38 86.48 90.26 91.37 88.74 83.55 90.35 86.04 91.72 88.92 90.38 88.51 85.55 89.35 87.91 92.27 85.45 89.27 85.41 88.65 91.45 85.57 87.65 92.02 91.87 86.95 85.08 88.30 88.51 92.38 84.90 92.77 87.06 87.54 87.85 93.23 86.21 88.02 91.87 89.78 88.97 88.68 87.31 89.69 91.31 92.22 90.44 88.53 86.30 84.20 88.30 90.51 92.85 86.95 89.86 92.78 91.61 90.55 88.76 87.39 88.94 89.63 90.98 90.54 91.22 88.20 83.70 90.30 87.53 89.50 92.09 91.28 89.61 90.18 92.37 88.40 90.45 89.36 91.01 88.90 91.05 87.69 91.34 88.03 88.71 90.28 92.04 88.54 90.69 91.05 87.94 90.25 87.78 90.46 90.69 88.39 91.16 89.80 87.57 90.26 90.35 87.97 91.28 91.48 87.03 90.66 90.13 92.13 89.05 88.44 89.88 90.90 88.66 90.44 90.14 90.69 89.62 91.10 88.78 90.31 90.46 88.97 91.39 90.91 90.77 87.40 89.88 88.39 90.83 89.53 87.43 87.85 28, species 9 DSM 1 DSM purpuratum 1059T; 32, cohohdsia marismortui Ectothiorhodospira pedioforme okenii .2 0.047 0.124 04 9.8 04 8.0 03 9.2 01 8.8 81 8.9 21 8.9 95 8.7 90.29 89.77 89.58 87.89 92.10 88.99 88.16 89.28 90.17 90.32 90.30 89.90 90.46 90.08 90.44 5261T; 24, 10 DSM and 76T; .5 007 .5 9.8 40 9.6 10 9.5 02 8.3 21 9.3 23 8.5 83 8.2 74 8.9 68 8.5 69 85.36 86.93 87.15 86.82 86.29 87.46 88.22 88.34 84.55 92.36 92.93 92.12 89.93 90.21 92.35 91.02 92.86 94.08 93.28 0.053 0.047 0.054 DSM 1 2 3 14 13 12 11 evolutionary aohdsia abdelmalekii Halorhodospira 3771T; 6, DSM 0.040 DSM Thiorhodococcus Thiococcuspfennigii 169T; 16, 1591T; 2, 20, 3802T; 1 (%), Thiocapsa 48 9.5 32 9.3 18 9.3 04 8.4 08 9.1 17 8.9 75 8.3 68 8.7 69 8.3 60 86.38 86.02 85.83 86.90 87.47 86.84 87.43 87.52 83.79 91.71 92.01 90.88 89.04 90.44 91.13 91.85 92.43 93.28 93.75 94.84 89.63 Allochromatium vinosum Allochromatium and the values on the distances Marichromatium gracile Marichromatium 1, 00 9.4 01 8.7 90 9.8 97 9.8 07 8.0 42 8.1 59 8.9 52 8.2 85.60 85.32 85.23 85.69 85.98 86.61 84-24 89.90 90.70 91.28 89.75 93.28 89.02 87.87 90.18 91.24 90.08 15 Thiocystis violascens minor pendens DSM DSM 16 of DSM 4 1 presently recognized 17 DSM 226; 7, DSM 80T; 1 15 1 21 29, 18 lower 236T; 21, hoaoas halophila Thiohalocapsa 8T; loT; DSM 1 DSM cohohdsia haloalkaliphila Ectothiorhodospira DSM 25, 9 0 21 20 19 DSM left are 33, Escherichia 80T; Amoebobacterpurpureus aohdsia halophila Halorhodospira 1 68T; 12, 203T; K,,, 17, 3, Chromatiurn values for corrected multiple change base Allochromatium minutissimum Allochromatium Halochromatium Thiocystis gelatinosa coli; 22 26, DSM 3 4 5 6 27 26 25 24 23 cohohdsia shaposhnikovii Ectothiorhodospira 9254 62 species loT; DSM DSM 244T. DSM glycolicum 82-62 42 8.9 79 8.0 81 8.2 58 8.6 86.50 85.86 85.86 87.42 88.16 87.80 87.99 88.59 84.24 ATCC 8,

Downloaded from www.microbiologyresearch.orgDSM by with 4197T; 22, Thiocapsa roseopersicina Thiocapsa 58 8.4 56 8.8 47 8.4 40 84.86 84.06 84.44 84.74 85.18 85.69 85.34 85.89 IP: 134.245.215.185 5

1

2 1

On: Tue, 30 May 2017 10:29:44other

935T;

DSM DSM 1376T; DSM 18,

5T; Thiorhodovibrio winogradshyi Thiorhodovibrio 13, 30, phototrophic purple 28 1 1080T; Thiocystis Ectothiorhodospira by 29 the methods of Jukes 4, DSM 30 Halochromatium Allochromatium minor DSM 31 243T; 217T; DSM ...... 85.18 2 33 32 mobilis sulfur 178T; 85.33 83.73 Table 33 32 31 30 29 28 27 26 25 23 22 21 20 24 19 18 17 .3 017 0.129 0.127 0.139 0.133 0.136 0.151 0.131 0.143 0.124 0.123 0.138 0.177 0.184 0.076 0.080 0.082 0.096 0.095 0.075 0.084 0.084 0.115 0.133 0.122 0.136 0.131 0.142 0.126 0.118 0.090 0.106 .6 014 0.149 0.154 0.160 0.157 0.161 0.166 0,169 0.138 0.135 0.152 123 2 (cont.) .6 0.157 0.162 0.177 0.079 0.120 0.136 0.126 0.134 0,131 0,104 0.1 0.127 0.122 0.128 0.1 0.162 0.084 0.080 0.07 0.151 0.148 0.152 0.132 0.126 0.122 0,120 0.102 4 15 11 1 0.103 0,092 0,082 0.088 0.091 0.132 0,125 0.123 0.133 0,143 0.119 0,142 0.124 .6 0.159 0.165 0.153 0.170 0.164 0.164 0,160 0.148 0,150 0.153 0.155 0.150 0.149 0.178 0.186 0.101 .6 0.158 0.164 56 0.105 0.110 0.155 0.134 0.133 0.126 0.118 0.115 0.159 0.069 0.060 0.057 0.104 0.103 0.125 0.116 0.086 0.143 0.143 0.143 7 .9 0.110 0.099 0.112 0.093 0.132 0.110 0.070 0.119 0.119 0.129 0.132 0,108 .5 0.156 0.155 0.152 0.147 0.198 0.177 .0 0.116 0.105 .6 0.169 0.163 0.179 0.165 0,174 0,160 0,163 0.165 0.164 0.163 0.159 89 0,083 0.171 0,104 .6 010 0.119 0.130 0.160 0.137 0.120 0.127 0.155 0.183 0.163 0.179 0.191 0.088 0.079 0.084 0.097 0.151 0.102 0.095 0.105 0.134 0.094 0.094 0.095 0.125 0.086 0.094 0.086 0.136 .0 0.087 0.100 0.097 0.081 0.087 0.111 .8 0,144 0.183 0.155 0.150 0.153 0.181 0.157 0.146 0.153 0.182 0,170 0.137 0.133 0.130 0.162 0.145 0.124 0.132 0.168 .2 004 .6 0.080 0.069 0.074 0.125 0 11 10 0.111 .4 014 0.144 0.134 0.143 0.081 0.118 0.105 0,148 12 0.084 0.150 0,138 13 0.173 0.081 0.074 0.151 0.144 0.141 0.145 0.151 0.137 0. 0.127 0,083 0.108 0.105 0.08 0.096 0,075 14 I28 1 0.156 0.154 0.158 0.145 0.141 0.136 0.128 0.129 0.184 0.087 0.094 0.084 0.111 0.096 0.103 0.101 0.08 15 1 ~ 0.148 0.139 0.136 0.126 0,130 0.129 0.118 0.1 0.163 0.065 0.084 0.079 0.114 0.120 0.073 0.065 0.016 16 14 0.145 0.148 0.142 0.133 0.130 0.175 0.082 0.098 0.098 0.118 0.159 0.153 0.156 0.125 0.088 0.070 17 0.155 0.160 0,161 0,148 0.150 0.146 0.137 0.135 0.194 0.112 0.115 0.115 0.128 0,130 0.090 93.32 18 0.112 0.078 0.148 0.135 0,167 0,153 0,152 0.168 0.145 0.162 0.148 0.160 0.148 0.161 0.142 0.155 0.140 0.207 0,194 0.101 0.104 0.103 0.106 15 88.07 91.54 88.46 91.66 19 0.101 0.186 0.185 0.180 87.61 20 0.181 0.185 0.186 0.166 0.161 0.164 0.155 0.157 0.209 0.104 0.107 92.57 0.1 86.53 88.20 89.08 21 10 0.129 0.129 0.120 0.131 0.122 0,127 0,122 0.115 0.162 0.062 0.042 90.01 90.56 90.8 89.62 89.35 22 1 .3 0.132 0.130 .4 0.145 0.142 03 90.58 90.39 90.25 90.12 .2 0.118 0.120 0.114 0.114 0.178 0.152 0.066 94.08 95.96 .3 0.143 0.132 0.140 0.134 0,128 0.137 0.127 93 89.63 89.32 92.22 90.77 97 90.32 89.75 23 93.65 0,136 24 0.179 0.212 0.199 0.201 0.198 0.208 0,190 0.180 84.18 86.22 85.42 8 8 82.92 82.88 84.37 1.78 1.88 25 0,129 0.121 0.112 0.122 0.117 0.044 0.042 0.035 0.031 0.044 0.045 0.0 40 83.97 84.07 89.04 89.45 88.91 89.40 88.78 89.38 85.97 85.82 85.52 86.02 87.07 87.24 87.47 87.65 Downloaded from 87.81 www.microbiologyresearch.org88.04 by 6 27 26

13 IP: 134.245.215.185

0.130 98.75 On: Tue, 30 May 2017 10:29:44 0.034 96.98 95.66 .4 0.043 0.048 96.59 95.72 .3 0.128 0.136 .3 0.131 0.137 0.129 0.143

83 88.76 88.33 80 88.33 88.08 32 81.86 83.20 87.49 87.87

71 86.56 87.10 67 86.43 86.70 65 86.60 86.59 56 85.47 85.62 53 85.48 85.30 8 29 28 96.72 ~ 0.143 95.84 95.32 95.75 95.92 0.1 0.1 82.59 87.57 88.24 87.94 85.10 84.95 86.85 86.60 86.82 30 15 16 0.063 0.01 89.23 88.26 87.55 88.76 89.14 82.39 87.22 87.72 88.91 83.52 83.96 86.20 85.49 85.92 31 8 0.067 93.92 98.26 93 86.98 89.36 87.98 88.19 87.44 86.97 88.08 88.80 88.18 89.61 81.55 82.50 86.83 86.97 87.04 87.89 81 88.15 88.17 83.95 83.63 83.55 83.62 85.01 86.13 86.03 85.61 85.64 86.15 32 93.60 33 J. F. Imhoff, J. Suling and R. Petri

Thiohnlocapsa halophila DSM 62Id Thiococcuspfmnig'i DSM226 Halochmmatium salexigens DSM 4395' Halochmmatium glycolicum DSM 11 08d I' Isochivmatium buderi DSM I7# Rhabdochromatium marinum DSM526Ir Thiorhodovibrio winograhkyi DSM 670P

Mnichromatium gracile DSM203' Thermochromatium tepidum DSM 3 771' Allochromatium warrningii DSM 173' Allochivmatium minutimimum DSM 1376'

Chivmatium okenii DSM I6f - Thiocystis violacea DSM 207' - Thiocystis minor DSM I781

Ectothiorhodospira haloalkaliphila ATCC SI 935' Ectothiorhodospira marismortui DSM 41 80' Ectothiorhodospira vacuolata DSM2III' IEctothiorhodospira shaposhnikovii DSM243' I Escherichia coli I I 0.05 K,,,

Fig. 1. Phylogenetic tree showing the relationships of the investigated type strains of Chromatiurn species and related purple sulfur bacteria. Derived from the distance matrix (Table 2) and calculated as indicated in Methods.

~

isolates from coastal environments do grow in the Morphological properties presence of low concentrations of salt but do not The principal classification schemes of require salt for growth (R. Guyoneaud & P. Caumette, Chromatiaceae personal communication). genera use cell form, motility by flagella and presence of gas vesicles as key properties (Imhoff & Truper, & DISCUSSION 1982;Pfennig Truper, 1974, 1989). Though it would be an exaggeration to state that these properties have The available 16s rDNA sequence information of no taxonomic meaning, it is clear that they are Ectothiorhodospiraceae (Imhoff & Suling, 1996) and insufficient taxonomic characteristics and not sufficient Chromatiaceae species (DeWeerd et al., 1990 ; to decide on the assignment of bacteria to a particular Wahlund et al., 1991; Caumette et al., 1997; genus. Non-motile spherical purple sulfur bacteria Guyoneaud et al., 1997, 1998; and this work) support without gas vesicles, for example, had been united the separation of these two families (Imhoff, 1984a) within the genus Thiocapsa. However, genetic relation- and at the same time demonstrate the inconsistency of ships indicate that two of the three known species the present taxonomic classification of the (Thiocapsa halophila and Thiocapsa pfennigii) are not Chromatiaceae with their genetic relationships. Most at all related to the type species of this genus, which is of the morphological and physiological properties, Thiocapsa roseopersicina. On the other hand, some of upon which the current taxonomy is based, have to be the spherical cells containing gas vesicles (former regarded as being of minor importance in support of a Amoebobacter species) are now also recognized as genetically oriented taxonomic classification, while members of this genus, indicating that the presence of others, so far much less considered properties, sup- gas vesicles should no longer be a genus determinative posedly are of higher significance in this regard. The property either. Nonetheless, one of the major phylo- differential characteristics of the genera and species of genetic clusters of Chromatiaceae is constituted out of the Chromatiaceae are summarized in Table 3, and are spherical cells without flagellar movement and with or discussed below. without gas vesicles (Fig. 1 ; Guyoneaud et al., 1998). Downloaded from www.microbiologyresearch.org by 1134 IP: 134.245.215.185 International Journal of Systematic Bacteriology 48 On: Tue, 30 May 2017 10:29:44 Phylogenetic taxonomy of Chrongatiaceae ~ ._

Table 3. Differential characteristics of genera and species of the Chromatiaceae family Genus and new Motile Gas Cell form Optimum Vitamins G + C Chemoauto- species name vesicles and size salinity $8) required content trophy ( rtm) (%) (mol %)

Halochroma tium Yes No Rod

Haloc*iironiatium salexigens 2.0-2.5 10 20-30 Bl2 64.6 Yes Halochromatiurn glycolicum 0.8-1.0 2.5-6.0 3&35 None 66-3 Yes Thiohalocupsa No No Sphere

Thiohaloc upsu haloph ila 1.5-2'5 5-7 20-30 BE 65.9-66-6 Yes Thiococcus No No Sphere TlZiocwcwiJ pjennigii 1.2-1.5 1-2 20-3 5 None 694-69.9 No Rhubdochromatium Yes No Rod Riiabck,c.iiromatium marinum 1.5-1.7 1 '5-5 30 None 60.4 No Isochromatium Yes No Rod

Isociirom~rtiuni buderi 3.54.5 1-3 25-30 Bl2 62'2-62.8 No Thiorhodococcus Yes No Sphere Thioriiotlococcus minor 1 *0-2*0 2 30-35 None 66.9 Yes Marichromatium Yes No Rod Mariciirotnatium gracile 1'0- 1* 3 2-3 30-35 None 68.9-70.4 Yes Mariciiromatiurn purpuratum 1.2-1.7 2-7 25-30 0 68.9 0 Thiorhodovibrio Yes No Spiral Thioriioclovibrio \iinograd,skyi 0.8-1.4 2-3 33 None 6 1*O-62.4 Yes A llochromatium Yes No Rod A Ilo c h so n icr t iuin v in osum 2.0 0 30-35 None 6 1*3-66.3 Yes A llochr on ici t ium minu t issimum 1.0-1.2 None 30-3 5 None 63.7 Yes

Allochroniatium warmingii 3.5-4.0 None 25-30 Bl2 55.1-60.2 No Thermochromatium Yes No Rod Themo cii roma t ium tepidum 1.0-2.0 None 48-50 None 61.5 No Chromatium Yes No Rod I Ciiromcr t iiin okenii 4.5-600 None 25-30 Bl2 48.0-50.0 No Cliromcrtium rceissei 3.54-0 None 25-30 Bl2 48.0-50.0 No Thiocysti.+ Yes No Sphere/rod 3-0 None 30 None 61.3 Yes 2.0 None 30 None 62.2 Yes Thiocj3.5ti.\ riolacea 2.5-3.5 0 25-35 None 62'8-67.9 Yes Tiiiocj9sti.\ violascens 2.0 0 30-35 None 61.8-64'3 Yes Thiocapsa No No Sphere Thioccrpbtr roseopevsicina 1.2-3.0 0 20-3 5 None 6 3.3-66.3 Yes

Tiiioctrpw rosea 2.0-3.0 None 20-35 42 64.3 Yes Thiocapstr pendens 1.5-2.5 None 20-35 Bl2 65.3 No Thiolamprovum No Yes Sphere

Thiolampsovumpediofovme 2.0 None 37 (I3121 65-5 Yes Amoebobacter No Yes Sphere Amoebohricter purpureus 1.9-2.3 None 23-25 0 63.5 Yes Thiodicty on No Yes Rod Tliiodictjvn elegans I '5-2.0 None 20-25 None 65.3 No Tiiiodic.ijv)n bacillosum 1.5-2.0 None 20-30 None 66.3 No Lamprobucter Yes Yes Rod

Lamprohrrcter modestohalophilus 2.0-2.5 1-2 25-27 B12 64.0 Yes Lamprocystis Yes Yes Sphere Lamproc~!~srisroseopersicina 3.0-3.5 None 20-30 None 63-8 No Thiospirillum Yes No Spiral

Thiospiriiluin jenense 2.54.5 None 20-25 Bl2 45.5 No Thiopediu No Yes Sphere

Tliiopecr'icr sosea 1-0-2.0 None 20 Bl2 62'5-63.5 No

~. Downloaded from www.microbiologyresearch.org by International Journal of Systematic Bacteriology 48 IP: 134.245.215.185 1135 On: Tue, 30 May 2017 10:29:44 J. F. Imhoff, J. Suling and R. Petri

Physiological properties taxonomic criterion. The salt requirement apparently is of general taxonomic importance in phototrophic Physiological properties always have been important bacteria and already has been considered in the characteristics in taxonomic considerations, although taxonomy of purple non-sulfur bacteria and their taxonomic significance is ambiguous. On the one Ecto- thiorhodospiraceae. It proved to be of relevance to hand, physiological properties of the Chromatiaceae distinguish the genera and are quite conservative in regard to the use of sulfur Halorlzodospira Ecto- (Imhoff & Suling, 1996), sources and the restricted use of organic carbon thiorhodospira Rhodovulum and Rhodobacter (Hiraishi & Ueda, 1994) and also sources. All species are able to grow photo- genera of the spiral-shaped purple non-sulfur autotrophically under anoxic conditions in the light a- Proteobacteria, the former Rhodospirillum species using sulfide or elemental sulfur as an electron donor. (Imhoff 1998). Furthermore, phylogenetic On the other hand, we distinguish two major physio- et al., relationships of the Chlorobiaceae indicate that salt logical groups, versatile and specialized species. The relationships may be a useful criterion also for these specialized species depend on strictly anoxic conditions bacteria (Overmann & Tuschak, 1997). and are obligately phototrophic. Sulfide is required, thiosulfate and hydrogen are not used as electron Genetic relationships of the Chromatiaceae (Fig. 1, donors. Only acetate and pyruvate (or propionate) are Table 2) revealed the obvious divergence of freshwater photoassimilated in the presence of sulfide and CO,. and saltwater bacteria. In particular the so far known These bacteria do not grow with organic electron genera Chromatium and Thiocapsa have repre- donors and chemotrophic growth is not possible. None sentatives in both of these phylogenetic branches. As a of these species assimilates sulfate as a sulfur source. consequence thereof, the salt relation should be con- Among these species are Chromatiurn okerzii, sidered as property for characterization of ' genetically Chromatiurn weissei, Chronzatiurn wwrmingii, defined ' genera. Thus, both the genetic relationships Chroulzat iurn buderi, Thiospirillum jenense and and the salt responses enable differentiation of the Thiocapsu pfennigii. The versatile species photo- halophilic Chromatiurn species C'lzromatiunz salexigem assimilate a greater number of organic substrates. and Chromatium glycolicum from the marine species Most of them are able to grow with sulfate and in the Chromatiurn gracile and Chromatiunz purpuratum, and absence of reduced sulfur sources with organic sub- from the freshwater species of Chromatium vinosurpi strates as electron donors for . Some and relatives. Although salt relationships obviously species even grow as chemoautotrophs, and a few are important properties for the taxonomic charac- species can grow chemoheterotrophically in addition terization of Clzromatiaceae, in a few species the (Gorlenko, 1974; Kondratieva et al., 1976; Kampf & congruence between their phylogenetic position and Pfennig, 1980, 1986). Among these species are several their salt response requires further attention. Species small-celled Chromatium species, Thiocystis violacea, such as Chomatium vinosum and Thiocapsa Thiocapsa roseopersicina, Thiocapsa rosea (formerly roseopersicina have been reported to be found in Amoebobacter roseus), Thiocapsa pendens (formerly freshwater, brackish water and marine habitats. In Amoebobacter pendens) and modesto- regard to Thiocapsa roseopersicina, sequence relation- halop h ilus. ships indicate that a number of strains isolated from the marine environment and assigned to this species Although some specialized species, such as could be distinguished at the species level (Guyoneaud Chromatiurn okenii, Chromatiurn buderi and Thiocapsa et al., 1998). These bacteria behave indifferently to low pfennigii form well-separated genetic lines, others like salt concentrations and may be poorly adapted to the Chromatiurn warmingii are moderately related to the marine environment, being halotolerant but not halo- versatile Chromatium vinosum, justifying their philic. In regard to Chromatiurn vinosum the situation inclusion into one and the same genus. Therefore, at is unclear at present. Large differences in the G+C present the versatility versus specialization of the content of the DNA of strains assigned to this species species appears not to be a good taxonomic criterion. (61 -3-66-3 mol YO)may indicate genetic heterogeneity. So far, clear proof of identity of various isolates from Salt relationships marine habitats assigned to Chromatium vinosum on the species level is lacking. Further studies will be Though salt dependence has been used already in the needed to determine whether differences in the G + C past to distinguish between species and even genera, content are correlated with the occurrence of these the phylogenetic and taxonomic importance of this bacteria in marine or freshwater environments. property has so far not been fully recognized. Major phylogenetic branches within the Chromatiaceae can DNA base ratio be distinguished on the basis of their salt requirement (Fig. 1, Table 2), one exclusively containing marine The DNA base ratio, expressed as the G+C content, and halophilic species, the other primarily freshwater which always has been regarded as a taxonomically isolates. This implies principally that a separate evol- important parameter, is gaining new attention in view utionary development has occurred in the marine and of a genetically oriented taxonomic system. It has been in the freshwater environments and that therefore the found to be highly variable in some of the known salt response should be considered as an important Chromatiaceae genera (e.g. values from 48.0 to Downloaded from www.microbiologyresearch.org by International Journal Systematic Bacteriology 48 1136 IP: 134.245.215.185 of On: Tue, 30 May 2017 10:29:44 Phylogenetic taxonomy of Chromatiaceae

70.4 mol (YOG + C in the genus Chronzatiunz).However, groups of species (J. F. Imhoff, unpublished results). it is assumed that to a large extent the intrageneric and For example, Clzromatium vinosum and Chromatium also the intraspecies variability represents the non- warmingii, two species of the proposed new genus phylogenetic nature of the current taxonomic system Allochromatium, contain the same lipids. Their lipid and misclassification of strains, respectively. A more composition is different from a second group of natural system would be expected to group bacteria species, the proposed members of the genus Thiocystis together that have much less variation in their G + C (Chromatium minus, Chromatium violascens and the content. Indeed, genetically closely related species of two known Tlziocystis species), which also show the Chroniatiaceae in general have a quite narrow uniformity in this property. In contrast, the lipid range of their G + C content, which is approximately composition of Thiocapsa pfennigii is different from all 63-66 mol YOin the Thiocapsa/Amoebobacter cluster, other so far analysed phototrophic purple bacteria (J. between 6 1-64 mol YOin the proposed Thiocystis group F. Imhoff, unpublished results). Thus, the pattern of (with the exception of a few strains of Thiocystis polar lipid composition, as far as known, is congruent violaceu), at 69-70 mol YO in the proposed with the sequence-based similarity of these bacteria Marichromatium species and at 64-66 mol% in the and may well turn out to be a significant property to proposed Halochromatium species. In the true distinguish genetically related groups of Chromcitium species it is as low as 48-50 mol YO.A few Clzromatinceae in the future. More analytical data are species. Chromatium vinosum (61 -3-66-3 mol YO), required to support this concept. Chromutiurn warmingii (55.1-60.2 mol YO), and Despite these first attempts to define phenotypic Thiocj7.sti.s violacea (62.8-67.9 mol YO) have properties that are congruent with the genetic surprisingly high intraspecies variation of their G + C relationships of the purple sulfur bacteria, the search content. Further studies are needed to clarify whether for more of such properties must be continued and analytical problems have caused these variations or those properties that are of relevance must be analysed misclassification of some strains is indicated. Variation in all known species. The differentiation of the at the intraspecies level of more than 5 and up to Chromatiaceae species on the basis of some diagnostic 10 mol YOG + C content of the DNA is by far beyond properties is shown in Table 3. an acceptable level of natural variation within bacteria as closely related as to justify their classification into Proposed taxonomic changes one and the same species. On the basis of their genetic relationships and in Temperature response consideration of the preceding discussion on the relevance of various phenotypic properties, the fol- The temperature response of many species is not well- lowing taxonomic changes are proposed : resolved and in most cases probably of minor significance for taxonomic purposes. However, as far (1) Chromatium minus and Chromatium violascens are as the temperature extremes are concerned, specific most closely related to Thiocystis gelatinosa, but adaptation mechanisms are involved, specific environ- different from this bacterium at the species level. mental niches are inhabited and a different evolution- Therefore, we propose to transfer these species to the ary background can be expected. Therefore, it is very genus Tlziocystis with the new names Thiocystis minor well indicated to taxonomically consider the successful comb. nov. and Thiocystis violascens comb. nov., adaptation to the extremes of temperature. The only respectively. species of the Chromatiaceae so far known, which (2) Chromatium vinosum, Chromatium minutissimum exhibits a very high optimum temperature response is and Chromatium tvarnzingii are closely related species Chronzntium tepidum (optimum at 48-50 "C), which that should be placed into a new genus, because of also is genetically distinct from other species within the their genetic distance from Chromatium okenii. Be- cluster of freshwater species. cause Chromatium vinosum is among the best-studied purple sulfur bacteria and the name Chromatium is Molecular parameters very much associated with this species, we propose the Though only scattered information is available on name Allochromatium gen. nov. ('the other fatty acid and quinone composition of Chromatiaceae, Chromatium') for the new genus, with Allochromatium it appears that these components, which are well- vinosum comb. nov. as the type species and accepted tools for identification and taxonomic Allochromatium minutissimum comb. nov. and differentiation of phototrophic bacteria (Imhoff, Allochromatium warmingii comb. nov. as additional 1984b, 1991 ; Imhoff & Bias-Tmhoff, 1995; Thiemann species of the genus. & Imhoff, 1996), show minor variation and are of low (3) Because Chromatium tepidum quite obviously can significance for taxonomic differentiation within the not be retained within the genus Chromatium and it is Clzromcrtiliceae (Imhoff & Bias-Imhoff, 1995). How- neither specifically related to Tlziocystis nor to ever, the polar lipid composition, that has been Allochromatium, it should also be placed in a new analysed so far only in several freshwater species, genus. The 16s rDNA sequence data justify this despite surprisingly similar patterns in genetically conclusion. Chromatium tepidum is the only thermo- related species, shows significant variation between philic isolate of the purple sulfur bacteria described so Downloaded from www.microbiologyresearch.org by International Journal of Systematic Bacteriology 48 1137 IP: 134.245.215.185 On: Tue, 30 May 2017 10:29:44 J. F. Tmhoff, J. Suling and R. Petri far. Although it is not well-established whether and to species of this branch to a degree that suggests its what extent the temperature response is of phylo- recognition as a separate genus for which the name genetic and taxonomic significance, we regard the Isochromatium is proposed. Chromatium buderi is thermophilic nature and high temperature optimum of transferred to this new genus as Isochromatium huderi this species as a valid criterion for genus differentiation comb. nov. and propose Thermochromatium gen. nov. as the new genus name and Thermochromatium tepidum comb. Emended description of the genus Chromatiurn Perty nov. as the new name for this species. 1852, 174AL (4) Chromatium salexigens and Chromatium Chromatiurn (Chro.ma'ti.um. Gr. n. chroma colour ; glycolicum shall be transferred to the new genus M.L. neut. n. Chromatium the one which is coloured). Halochromatium gen. nov. because of their extended salt dependence and salt tolerance, which is the highest Cells are straight or slightly curved rods, more than 3 pm wide, motile by a polar tuft of flagella. Cells within the Chromatiaceae, with Halochromatium comb. nov. as the type species and occur singly or in pairs. Multiply by binary fission. salexigens Halo- Gram-negative. Contain internal photosynthetic chromatiuni glycolicum comb. nov. as an additional species. membrane systems of vesicular type in which the photosynthetic pigments bacteriochlorophyll a and (5) Chromatium gracile and Chromatium purpuratum carotenoids are located. Obligately phototrophic, are transferred to the new genus Marichromatium. The strictly anaerobic, require sulfide-reduced media. name is proposed according to the marine origin and Under anoxic conditions capable of photo- the moderate salt dependence that is characteristically lithoautotrophic growth with sulfide or elemental found in typical marine bacteria. The new names sulfur as electron donor. Elemental sulfur is formed as Marichrornatium gracile comb. nov. and an intermediate oxidation product and stored in the Marichromatiuin purpuratum comb. nov. are proposed form of highly refractile globules inside the cells. for these two species. Marichromatium gracile is the Sulfate is the ultimate oxidation product. In the proposed type species. presence of sulfide and bicarbonate, able to photo- (6) By its intracytoplasmic membrane system and its assimilate only a few simple organic compounds such pigment composition, Thiocapsa pfennigii is unique as acetate and pyruvate. Assimilatory sulfate reduction among the family Chromatiaceae (Eimhjellen et al., is lacking. Vitamin B,, is required. Mesophilic with 1967; Eimhjellen, 1970). Certainly, it can no longer be optimal growth temperatures of 20-30 "C, freshwater regarded as a species of the genus Thiocapsa, because it bacteria without a distinct requirement for salt. Habi- is significantly different from this genus on the basis of tat: ditches, ponds and lakes with stagnant freshwater the available genetic sequence information and belongs containing hydrogen sulfide and exposed to light. The to the cluster of the halophilic Chromatiaceae species. G + C content of the DNA is 48.0-50-0 mol% (Bd). Its phenotypic traits together with the results on its Type species is Chromatiurn okenii (Ehrenberg 1838, genetic relationships to other Chromutiaceae led us to Perty 1852, 174AL). Most likely also Chromatiurn consider this species as a separate genus. According to weissei is a species of this genus. Rule 33c of the International Code of Nomenclature of Bacteria (Lapage et al., 1992), we propose to revive Emended description of the genus Thiocystis the genus name Thiococcus Eimhjellen et al. 1967 and Winogradsky 1888, 60AL to transfer Thiocapsa pfennigii to Thiococcus pjennigii Thiocystis (Thi.o.cys'tis. Gr. n. thios sulfur; Gr. n. gen. nom. rev., comb. nov. The name Thiococcus, cystis the bladder, a bag; M.L. fem. n. Thiocystis sulfur originally used by Eimhjellen et al. (1967) to describe this bacterium, was not validly published and is bag). illegitimate and therefore was not included into the Cells are rod-shaped or ovoid to spherical, before cell Approved Lists of Bacterial Names. division often diplococcus-shaped. Cells occur singly or in pairs, may grow in irregular aggregates sur- (7) Phylogenetically, the bacterium described as rounded by slime. Multiply by binary fission, motile by Thiocapsa Iialophila is not related to the genus means of flagella. Gram-negative. Contains internal Thiocapsa and is most similar on the basis of its 16s photosynthetic membrane systems of vesicular type in rDNA sequence to Thiocupsa pfennigii, Chromatiurn which the photosynthetic pigments bacteriochloro- salexigens and Chromatium glycolicum (Fig. 1). Be- phyll a and carotenoids are located. Under anoxic cause of significant genotypic and phenotypic proper- conditions, cells are capable of photolithoautotrophic ties to these bacteria, it should be recognized as a growth with sulfide or sulfur as electron donor, separate and new genus. Thiohalocapsa gen. nov. is thiosulfate may also be used. During sulfide oxidation, proposed as the new genus name and Thiocapsa globules of elemental sulfur are transiently stored halophila is transferred to the new genus as inside the cells. The final oxidation product is sulfate. Tliiohalocapsa halophilu comb. nov. In the presence of sulfide and bicarbonate, several (8) Chromatium buderi is a marine species of the simple organic substrates are photoassimilated. Clzromatiaceae. However, according to its 16s rDNA Facultatively chemoautotrophic or mixotrophic under sequence it is dissimilar to any of the other described micro-oxic to oxic conditions. Mesophilic bacteria Downloaded from www.microbiologyresearch.org by International Journal of Systematic Bacteriology 48 1138 IP: 134.245.215.185 On: Tue, 30 May 2017 10:29:44 Phylogenetic taxonomy of Chrornatiaceae with good growth at 25-35 “C, pH 6.5-7.6. Principally Description of Thiococcus pfennigii comb. nov. freshwater bacteria, several of which may be found also in brackish and marine environments. Habitat: Thiococcus pfennigii (Thiocapsa pfennigii Eimhjellen anoxic sulfide-containing water and mud of freshwater 1970, 193AL)(pfen.nig’i.i. M.L. gen. n. pfennigii of ponds and lakes as well as brackish water and marine Pfennig; named after Norbert Pfennig, a German environments exposed to light. The G+ C content of microbiologist). The description is the same as that for the DNA is 61-3-67.9 mol% (Bd). Type species is Thiocapsa pfennigii (Eimhjellen, 1970 ; Pfennig & Truper, 1989). Thiocystis violacea Winogradsky 1888, 65AL.Other species are Thiocystis gelatinosa, Thiocystis minor and Thiocys t is violascens . Description of Thiohalocapsa gen. nov. Thiohalocapsa (Thi.o.ha.lo.cap’sa. Gr. n. thios sulfur; Gr. gen. n. halos of the salt; L. fem. n. capsa capsule; Description of Thiocystis minor comb. nov. M.L. fem. n. Thiohalocapsa the sulfur capsule of the salt). Thio cy s t is minor ( Chroma t ium min us Wino gradsk y 1888, 99AL) (mi’nor. L. fem. comp. adj. minor less, Cells are spherical, non-motile ; multiply by binary smaller). The description is the same as that for fission. Individual cell surrounded by a strong slime Chromatium minus (Winogradsky, 1888 ; Pfennig & capsule ; cell aggregates may be formed. Gram-nega- Truper, 1989). tive. Internal photosynthetic membrane system of vesicular type containing the photosynthetic pigments bacteriochlorophyll a and carotenoids. Under anoxic Description of Thiocystis violascens corn b. nov. conditions capable of photoautotrophic growth with sulfide, elemental sulfur and possibly other electron Thio cys t is v iolascens ( Chroma t ium violascens Pert y donors. During oxidation of sulfide, globules of 1852, 1 7424L) (vi.o.las’cens. L. part. violascens elemental sulfur are transiently stored inside the cells, becoming violet). The description is the same as that final oxidation product is sulfate. Assimilatory sulfate for Chromatium violascens (Perty, 1852 ; Pfennig 8z reduction lacking. Chemoautotrophic or chemo- Truper, 1989). organotrophic growth possible under micro-oxic to oxic conditions in the dark. Vitamin B,, is required. Mesophilic bacteria with good growth at 20-30 OC, Description of Thiococcus gen. nom. rev. pH 6.5-7.5. Elevated concentrations of sodium chlor- ide are required for growth. Habitat : sulfide-contain- Thiococcus (Thi.o.coc’cus. Gr. n. thios sulfur; L. masc. ing waters and sediments of saltern and other coastal n. coccus sphere; M.L. masc. n. Thiococcus sphere habitats with elevated salt concentrations and exposed with sulfur). The genus name Thiococcus was proposed to light. The G+C content of the DNA is 65.9- for this bacterium by Eimhjellen et (11. (1967). How- 66.6 mol % (Bd). Type species is Thiohalocapsa ever, this name has never been validly published and ha loph ila . the organism was validly named Thiocapsa pfennigii (Eimhjellen, 1970 ; Approved Lists of Bacterial Names, Description of Thiohalocapsa halophila comb. nov. 1980). The name Thiococcus is revived according to Rule 33c of the International Code of Nomenclature Thiohalocapsa halophila (Thiocapsa halophila of Bacteria (Lapage et al., 1992). Caumette et al. 1991) (ha.lo’phi.la. Gr. n. hals salt; Gr. adj. philos loving; M.L. fem. adj. halophila salt- Cells are spherical with diplococcus-shaped division loving). The description is the same as that for stages. Multiply by binary fission. Gram-negative. Thiocapsa halophila (Caumette et al., 199 I). Internal photosynthetic membrane system consisting of bundles of ribbonlike branched tubes continuous Description of Halochromatium gen. nov. with the cytoplasmic membrane. Individual cells colourless. colour of cell suspensions yellowish to Halochrowlatium (Ha.lo.chro.ma’ti.um. Gr. gen. n. orange-brown. Photosynthetic pigments are bacterio- halos of the salt; Chromatium a genus name; M.L. chlorophyll b and carotenoids. Obligately photo- neut. n. Halochromatium the Chromatium of the salt). trophic and strictly anaerobic. Capable of photo- Cells are straight to slightly curved rods, multiply by lithoautotrophic growth with sulfide and sulfur as binary fission; motile by polar flagella. Cells occur electron donor. Elemental sulfur globules are tran- singly or in pairs. Gram-negative. Contain internal siently stored inside the cells, final oxidation product is photosynthetic membrane systems of vesicular type in sulfate. In the presence of sulfide and bicarbonate which the photosynthetic pigments bacterio- organic substrates are photoassimilated. Good growth chlorophyll a and carotenoids are located. Under at 20-35 OC, pH 6.5-7.5. Salt is required for optimum anoxic conditions in the light capable of photo- growth. Habitat: brackish and marine waters and lithoautotrophic growth with sulfide or elemental sediments containing hydrogen sulfide and exposed to sulfur as electron donor. Elemental sulfur is formed as the light. The G+C content of the DNA is 69.4- an intermediate oxidation product and stored in the 69.9 mol YO(Bd). Type species is Thiococcus pfennigii. form of highly refractile globules inside the cells. Downloaded from www.microbiologyresearch.org by lnterna tional Journal of Systema tic Bacteriology 48 1139 IP: 134.245.215.185 On: Tue, 30 May 2017 10:29:44 J. F. Imhoff, J. Wing and R. Petri

Sulfate is the ultimate oxidation product. Hydrogen taining hydrogen sulfide and exposed to light, marine may be used as electron donor. In the presence of sponges and other marine invertebrates. The G+C sulfide and bicarbonate organic substrates can be content of the DNA is 68-9-70.4 mol YO(Bd). Type photoassimilated. Chemolithotrophic and chemo- species is Marichromatium gracile. An additional organotrophic under micro-oxic conditions in the species is Marichromatium purpuratum. dark. Not capable of assimilatory sulfate reduction. Mesophilic with optimal growth temperatures of Description of Marichromatium gracile comb. nov. 20-35 “C, optimum pH 7.2-7.6. Elevated salt concen- trations are required for growth. Habitat: reduced Marichromatium gracile (Chromatium gracile sediments in salinas, microbial mats in hypersaline Strzeszewski 1913, 321”IJ) (gra’ci.le. L. neut. adj. environments, and coastal lagoons with elevated salt gracile thin, slender). The description is the same as concentrations, which are exposed to light. The G + C that for Chromatium gracile (Strzeszewski, 1913 ; content of the DNA is 64.666.3 mol% (Bd, HPLC). Pfennig & Truper, 1989). Type species is Halochromatium salexigens. An ad- ditional species is Halochromatiurn glycolicum. Description of Marichromatium purpura tum comb. nov. Description of Halochromatium salexigens com b. nov. Mar ichr oma t ium purpurat um (Chromatium purpuratum Imhoff and Truper 1980b, 60 1’ ”) Hulochromatium salexigens (Chromatium sale..cigens (pur.pur.a’tum. L. neut. adj. purpuratum dressed in Caumette et al. 1988) (sal.ex’i.gens. L. n. sal salt; L. v. purple). The description is the same as that for exigo demanding, to demand; M.L. part. adj. Chromatiurn purpuratum (Imhoff & Truper, 1980a). salexigens salt-demanding). The description is the same as that for Chromatiuni salexigens (Caumette et ul., 1988). Description of Thermochromatium gen. nov. Thermochromatium (Ther.mo.chro.ma’ti.um. Gr. adj. Desc ri pt i o n of Halochromatium glycolicum co m b. thermos hot; Chromatium a genus name; M.L. neut. n. nov. Thermochroma tium the hot Chromatium). Halochroma tium glycolicum (Chromatium glycolicum Cells are straight to slightly curved rods. Multiply by Caumette et al. 1997) (gly.co’li.cum. M.L. neut. adj. binary fission, motile most likely by polar flagella. glycolicum related to glycolate as a substrate). The Gram-negative. Contain internal photosynthetic description is the same as that for Chromatium membrane systems of vesicular type in which the glycolicum (Caumette et al., 1997). p ho t 0syn t he tic pigments bac teri ochlo ro p h yll a and carotenoids are located. Obligately phototrophic, Description of Marichromatium gen. nov. photolithoautotrophic growth with sulfide or elemen- tal sulfur as electron donor under anoxic conditions in Mar ichroma t ium (Ma.ri .chr 0.ma’ ti. um . L . gen. n. the light. Elemental sulfur is formed as an intermediate maris of the sea; Chromatium a genus name; M.L. oxidation product and stored inside the cells. Sulfate is neut. n. Mnrichromatium the Chromatium of the sea, the ultimate oxidation product. In the presence of the truly marine Chromatium). sulfide and bicarbonate, organic substrates can be Cells are straight to slightly curved rods, multiply by photoassimilated. Thermophilic bacteria with optimal binary fission, motile by polar flagella, occur singly or growth temperatures at 48-50 “C, pH optimum is 7.0. in pairs or may stick together and form clumps. Gram- Freshwater bacteria without a requirement for negative. Contain internal photosynthetic membrane significant levels of salt. Habitat : sulfide-containing systems of vesicular type in which the photosynthetic hot springs of neutral to alkaline pH. The G+C pigments bacteriochlorophyll a and carotenoids are content of the DNA is 60-63 mol Yo (Bd). Type species located. Capable of photolithoautotrophic growth is Thermochromatium tepidum. with sulfide or elemental sulfur as electron donor under anoxic conditions in the light. Elemental sulfur is formed as an intermediate oxidation product and Description of Thermochromatium tepidum corn b. stored in the form of highly refractile globules inside nov. the cells. Sulfate is the ultimate oxidation product. Thermochroma tium tepidum ( Chromatium tepiduni Molecular hydrogen may be used as electron donor. In Madigan 1986) (te’pi.dum. L. neut. adj. tepidunz the presence of sulfide and bicarbonate, organic lukewarm). The description is the same as that for substrates can be photoassimilated. Chemolitho- Chromatium tepidum (Madigan, 1986). trophic and chemo-organotrophic growth may be possible under micro-oxic conditions in the dark. Description of Allochromatium gen. nov. Mesophilic bacteria with optimal growth temperatures of 25-35 OC, pH 6.5-7.6. Optimum growth at salt Allochromatium (Al.lo.chro.ma’ti.um. Gr. adj. allos concentrations typical of marine bacteria. Habitat : the other; Chromntium a genus name; M.L. neut. n. anoxic marine sediments and stagnant waters con- Allochromatium the other Chromatium). Downloaded from www.microbiologyresearch.org by International Journal of Systematic Bacteriology 48 1140 IP: 134.245.215.185 On: Tue, 30 May 2017 10:29:44 Phylogenetic taxonomy of Chromatiaceae

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Cells are straight to slightly curved rods. Multiply by Isochrornatium the similar Chromatium, the bacterium binary fission, motile by polar flagella. Cells occur similar to Chromatium). singly or in pairs. Gram-negative. Contain internal photosynthetic membrane systems of vesicular type in Cells are straight to slightly curved rods, multiply by which the photosynthetic pigments bacteriochloro- binary fission, motile by a polar tuft of flagella. Gram- negative. Contain internal photosynthetic membrane phyll CI and carotenoids are located. Photo- lithoautotrophic growth with sulfide or elemental systems of vesicular type in which the photosynthetic sulfur as electron donor under anoxic conditions in the pigments bacteriochlorophyll a and carotenoids are light. Elemental sulfur is formed as an intermediate located. Obligately phototrophic and strictly anaer- oxidation product and stored in the form of highly obic. Under anoxic conditions in the light capable of refractile globules inside the cells. Sulfate is the photolithoautotrophic growth with sulfide or elemen- ultimate oxidation product. Molecular hydrogen may tal sulfur as electron donor. Elemental sulfur is formed be used as electron donor. In the presence of sulfide as an intermediate oxidation product and stored in the and bicarbonate organic substrates can be photo- form of highly refractile globules inside the cells. assimilated, potentially mixotrophic. Mesophilic bac- Sulfate is the ultimate oxidation product. In the teria with optimal growth temperatures of 25-35 “C, presence of sulfide and bicarbonate organic substrates pH range 6.5-7.6. No distinct requirement for salt, can be photoassimilated. Mesophilic bacteria with except for strains that originate from the marine and optimal growth temperatures of 25-35 OC, pH range brackish water environment and may tolerate or 6-5-7.6. Salt is required for growth at concentrations require low concentrations of salt. Habitat: ditches, typical for marine bacteria. Habitat: estuarine salt flats ponds and lakes with stagnant freshwater containing and salt marshes. The G+C content of the DNA is hydrogen sulfide and exposed to light; also sewage 62.2-62-8 mol YO(Bd). Type species is Isochromatium lagoons, estuaries and salt marshes. The G + C content buderi. of the DNA is 55.1-66-3 mol% (Bd). Type species is Allochronicrtiuni vinosum. Other species are Allo- Description of lsochromatium buderi corn b. nov. chroniutiurn minutissimum and Allochromatium lvarnz ingii. Isochromatium buderi (Chromatiurn buderi Triiper and Jannasch 1968, 364AL)(bu’deri M.L. gen. n. buderi of Description of Allochromatium vinosum corn b. nov. Buder; named after J. Buder, a German plant physi- ologist). The description is the same as that for Allochrormtium vinosum (Monas vinosa Ehrenberg Chromatium buderi (Truper & Jannasch, 1968). 1838, 1 1 ; Chromatium vinosum Winogradsky 1888, 99“L) (vi.no’sum. L. neut. adj. vinosum full of wine). The description is the same as that for Chromatium ACKNOWLEDGEMENTS (Winogradsky, 1888 ; Pfennig & Truper, vinosun-1 We would like to express our gratitude to Professor Dr H. 1989). G. Triiper (University of Bonn, Germany) for discussion on and proposal of new names and Professor Dr N. Pfennig Desc ri pt ion of Allochromatium min utissimum comb. (Uberlingen, Germany) for providing the original cultures nov. and introduction into the handling of these creatures, Allochromrtium min u t issimum (Chromat ium niinutissinium Winogradsky 1888, 1OOAL) REFERENCES (mi.nu.tis’si.mum. L. neut. sup. adj. minutissimum Ambler, R. P., Daniel, M., Hermoso, J., Meyer, T. E., Bartsch, R. G. very small, smallest). The description is the same as & Kamen, M. D. (1979). 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