This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nereng, K. S. Articles by Kaplan, S. Search for Related Content PubMed PubMed Citation Articles by Nereng, K. S. Articles by Kaplan, S.

 Previous Article  |  Next Article 

Journal of Bacteriology, March 1999, p. 1684-1688, Vol. 181, No. 5
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Genomic Complexity among Strains of the Facultative Photoheterotrophic Bacterium Rhodobacter sphaeroides

Department of Microbiology and Molecular Genetics, The University of Texas Health Science Center Medical School, Houston, Texas 77030

Received 25 September 1998/Accepted 21 December 1998

	ABSTRACT

Top Abstract Text References

Pulsed-field gel electrophoresis following the use of rare cutting restriction endonucleases together with Southern hybridization, using markers distributed on chromosomes I and II of Rhodobacter sphaeroides 2.4.1, has been used to examine approximately 25 strains of R. sphaeroides in an effort to assess the occurrence of genome complexity in these strains. The results suggest that genome complexity is widespread and is accompanied by substantial genomic heterogeneity.

	TEXT

Top Abstract Text References

In this study, we have examined approximately 25 strains of diverse origins, but otherwise classified as Rhodobacter sphaeroides. We have determined minimum genome size and overall macro-restriction pattern polymorphisms by pulsed-field gel electrophoresis (PFGE). The number of rrn operons present in each strain and their direction of transcription relative to each other were also determined. Gene probes diagnostic for chromosomes CI and CII (11, 22, 23, 27) of R. sphaeroides 2.4.1, together with the above-described results, have been used to assess the likelihood that these strains contain more than one chromosome, i.e., genome complexity. In a recent report Jumas-Bilak et al. (14) have surveyed genomic complexity in chromosome number amongst members of the -3 subgroup and related subgroups of the Proteobacteria. When several strains of Agrobacterium were examined all had complex genomes, whereas the same was not true for Brucella (14). This observation raises important questions and warrants an examination of the distribution of genome complexity amongst members of the R. sphaeroides group of organisms. Is strain R. sphaeroides 2.4.1 an isolated example, or is the existence of multiple chromosomes within R. sphaeroides more widespread?

Based upon previous work in this laboratory (27, 28), we slightly modified electrophoretic conditions required to optimally separate large, mid-size, and small AseI-generated DNA fragments derived from the strains listed in Table 1, which were grown as previously described (26, 29). From these data (Table 2) we were able to estimate the minimal genome sizes for each of the strains examined. Table 2 is presented so as to enable the reader to visually infer the DNA fragment distributions and to provide fragment sizes. The running conditions which were employed did not permit us to accurately estimate fragment sizes greater than 1,100 kb, thus the indication in some instances of >1,105 kb. Previous work identified which AseI fragments represent plasmid DNA in R. sphaeroides 2.4.1 (24, 29). However, we did not attempt to identify plasmid-derived AseI fragments in any of the other strains examined here, although earlier studies (8) indicate that the 110-kb replicon of strain 2.4.1 was the largest apparent plasmid observed.

View this table: [in this window] [in a new window]   TABLE 1.   Bacterial strains and plasmids

View this table: [in this window] [in a new window]   TABLE 2.   Summary of AseI fragment sizes and estimate of minimum genome size

In order to use the distribution of various marker genes (3, 4), namely hemA and rbcL on chromosome I and hemT and rbcR on chromosome II (Table 1) (22, 27, 28) of strain 2.4.1, as representative of the presence of two chromosomes in diverse strains of R. sphaeroides, all of the strains were subjected to PFGE following digestion with the intron-encoded restriction endonuclease I-CeuI (16). Nylon membranes derived from the TAFE gels were prepared as previously described (27), and these were probed at high stringency with radiolabeled (25) purified internal fragments of both the 23S and 16S rrn cistrons (Table 1) (7). In strain 2.4.1 one rrn operon is located on chromosome I and two rrn operons are located on chromosome II, 30 kb apart (7, 27). Sequence analysis confirmed the recognition site for I-CeuI to be present within each 23S cistron, and the 23S rrn probe encompassed this sequence. When 2.4.1 is treated with I-CeuI, the large chromosome is linearized to a 3-Mbp DNA fragment and the small chromosome yields a 0.87-Mbp fragment and a 30-kb fragment, as expected. Hybridization of 2.4.1 with the 23S and 16S rrn probes yields both the expected and identical numbers of signals, confirming the presence of three rrn operons transcribed in the same direction.

Table 3 provides a summary of this analysis. Several features are immediately obvious. The numbers of rrn operons varies from two to five depending upon the strain of R. sphaeroides. Those strains of R. sphaeroides showing fewer signals with the 16S rrn probe than with the 23S rrn probe indicate that one or more operons are transcribed in opposite directions relative to one another. Thus, unlike Escherichia coli (13), Salmonella typhimurium (13, 17), and Bacillus subtilis (12), which appear to possess a constant number of rrn operons, strains of R. sphaeroides are quite heterogeneous. However, Bacillus cereus may also be heterogeneous (2). This heterogeneity could reflect the existence of rrn operons on multiple linkage groups, which over time and through unequal crossing over give rise to variable numbers of such operons. Nonetheless, these observations raise interesting, and for the moment unique, questions regarding the derivation of each of these strains.

View this table: [in this window] [in a new window]   TABLE 3.   Summary of results of SoutherN hybridization analysis

Nearly all strains, with the exception of IL106, 28/5, 17024, and 17025, possess at least two large I-CeuI-generated fragments, as is the case for 2.4.1. Further, the distribution of hemA and rbcL on the large I-CeuI fragment is true for all strains with the exception of 28/5 even when we consider the existence of a heterologous rbcL-generated signal for strain 160 and the absence of a hemA signal in strain 33575. The distribution of hemT and rbcR for the most part is similar to that observed for 2.4.1 (17 of 23 strains), although somewhat greater heterogeneity in their distribution is present (6 of 23). Again 28/5 and 33575 stand out. In fact, if we also consider the AseI-generated restriction pattern, the data for strain 28/5 suggest that it may not be R. sphaeroides, at least by the criteria employed here. Likewise, the results observed for strain 33575 make any conclusions regarding it difficult at the present time. Like 2.4.1, strains 17024 and 17025 possess, in addition to the large 3.0-Mbp I-CeuI-generated DNA fragment, a much smaller 0.40-Mbp second fragment. However, the presence of five rrn operons in these strains could be responsible for the smaller size of the second I-CeuI-generated fragment, if we assume that approximately four of the five rrn operons are present on the smaller of the two linkage groups. Thus, by a combination of criteria, as described here, most strains of R. sphaeroides obtained from culture collections around the world appear to contain at least two chromosomes, using strain 2.4.1 as the prototype. Further, our findings that additional markers, e.g., groEL₁, groEL₂, rpoN₂, rpoN₁, etc., are distributed between the two chromosomes of strain 2.4.1 (23a) add to a growing list of representative genes which might be used to more accurately assess genome complexity within R. sphaeroides. However, only the actual determination of the complete physical maps of the genomes of each isolate reported here represents an iron-clad approach to answering the questions posed here.

Finally, an issue regarding strain identification in the published literature is worth addressing. Strains 14690, NCIMB 8253, and RS2 appear to be identical to 2.4.1. The absence of the 5-kb restriction fragment from the PFGE profile of NCIMB 8253 should not be considered a difference since this fragment is very often difficult to observe because it diffuses so readily in the gel. Strain RS2 has a 380-kb AseI fragment in place of the 410-kb fragment in 2.4.1. We have repeatedly observed that a 30-kb fragment can spontaneously excise from chromosome I of 2.4.1, reducing the 410-kb fragment to 380 kb (28, 29). Once removed, this fragment is lost from the genome. Strain 158, except for the presence of two rrn operons, is otherwise identical to 2.4.1 by the criteria applied here.

However, it should also be noted that some strains described here have been reported to be identical to 2.4.1, e.g., ATCC 17023 and an 8253 derivative obtained from R. Niederman some years ago. Although certainly similar, these strains are not identical to 2.4.1, and therefore we have arbitrarily designated the 2.4.1 strain in our laboratory as R. sphaeroides 2.4.1^T.

	ACKNOWLEDGMENTS

We acknowledge the contribution by M. Moore of historical information with regard to strains analyzed in this study, the scientific support of J. Zeilstra-Ryalls, and the patient computer support of C. Mackenzie and A. Simmons. We also wish to thank M. Choudhary for his assistance in the phylogenetic analyses.

This work was supported by a grant from the NIH (GM55481).

	FOOTNOTES

^* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, The University of Texas Health Science Center Medical School, 6431 Fannin, Houston, TX 77030. Phone: (713) 500-5502. Fax: (713) 500-5499. E-mail: skaplan{at}utmmg.med.uth.tmc.edu.

	REFERENCES

Top Abstract Text References

1.	American Type Culture Collection. 1991. Catalogue of bacteria and phages, 18th ed. American Type Culture Collection, Rockville Md.
2.	Carlson, C. R., and A.-B. Kolstj. 1994. A small (2.4Mb) Bacillus cereus chromosome corresponds to a conserved region of a larger (5.3Mb) Bacillus cereus chromosome. Mol. Microbiol. 13:161-169[Medline].
3.	Choudhary, M., C. Mackenzie, K. Nereng, E. Sodergren, G. Weinstock, and S. Kaplan. 1997. Low resolution sequencing of Rhodobacter sphaeroides 2.4.1T: chromosome II is a true chromosome. Microbiology 143:3085-3099[Abstract].
4.	Donohue, T. J., and S. Kaplan. 1992. Genetic techniques in Rhodospirillaceae. Methods Enzymol. 204:459-485.
5.	Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH. 1989. Catalogue of strains, 4th ed. Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Federal Republic of Germany.
6.	Dryden, S. C. 1992. Ph.D. thesis. University of Illinois, Urbana-Champaign.
7.	Dryden, S. C., and S. Kaplan. 1990. Localization and structural analysis of the ribosomal RNA operons of Rhodobacter sphaeroides. Nucleic Acids Res. 18:7267-7277[Abstract/Free Full Text].
8.	Fornari, C. S., M. Watkins, and S. Kaplan. 1984. Plasmid distribution and analysis in Rhodopseudomonas sphaeroides. Plasmid 11:39-47[Medline].
9.	Gest, H., M. W. Dits, and J. L. Favinger. 1983. Characterization of Rhodopseudomonas sphaeroides strain `cordata/81-1'. FEMS Microbiol. Lett. 17:321-325.
10.	Hallenbeck, P. L., and S. Kaplan. 1987. Cloning of the gene for phosphoribulokinase activity from Rhodobacter sphaeroides and its expression in Escherichia coli. J. Bacteriol. 169:3669-3678[Abstract/Free Full Text].
11.	Hallenbeck, P. L., R. Lerchen, P. Hessler, and S. Kaplan. 1990. Roles of CfxA, CfxB, and external electron acceptors in regulation of ribulose 1,5-biphosphate carboxylase/oxygenase expression in Rhodobacter sphaeroides. J. Bacteriol. 172:1736-1748[Abstract/Free Full Text].
12.	Jarvis, E. D., R. L. Widom, G. LaFauci, Y. Setoguchi, I. R. Richter, and R. Rudner. 1988. Chromosomal organization of rRNA operons in Bacillus subtilis. Genetics 120:625-635[Abstract/Free Full Text].
13.	Jinks-Robertson, S., and M. Nomura. 1987. Ribosomes and tRNA, p. 1358-1385. In Escherichia coli and Salmonella typhimurium, cellular and molecular biology, vol. 2. American Society for Molecular Biology, Washington, D.C.
14.	Jumas-Bilak, E., S. Michaux-Charachon, G. Bourg, M. Ramuz, and A. Allardet-Servent. 1998. Unconventional genomic organization in the alpha subgroup of the Proteobacteria. J. Bacteriol. 180:2749-2755[Abstract/Free Full Text].
15.	Lascelles, J. 1956. The synthesis of prophyrins and bacteriochlorophyll by cell suspensions of Rhodopseudomonas sphaeroides. Biochem. J. 62:78-93.
16.	Liu, S.-L., A. Hessel, and K. E. Sanderson. 1993. Genomic mapping with I-CeuI, an intron-encoded endonuclease specific for genes for ribosomal RNA, in Salmonella spp., Escherichia coli, and other bacteria. Proc. Natl. Acad. Sci. USA 90:6874-6878[Abstract/Free Full Text].
17.	Liu, S.-L., and K. E. Sanderson. 1995. I-CeuI reveals conservation of the genome of independent strains of Salmonella typhimurium. J. Bacteriol. 177:3355-3357[Abstract/Free Full Text].
18.	Meinhardt, S. W., P. J. Kiley, S. Kaplan, A. R. Crofts, and S. Harayama. 1985. Characterization of light-harvesting mutants of Rhodopseudomonas sphaeroides. I. Measurement of the efficiency of energy transfer from light harvesting complexes to the reaction center. Arch. Biochem. Biophys. 236:130-139[Medline].
19.	Muller, E. D., J. Chory, and S. Kaplan. 1985. Cloning and characterization of the gene product of the form II ribulose-1,5-bisphosphate carboxylase gene of Rhodopseudomonas sphaeroides. J. Bacteriol. 161:469-472[Abstract/Free Full Text].
20.	Nara, T., M. Misawa, and R. Okachi. August 1972. U.S. patent 3,682,777.
21.	National Collections of Industrial and Marine Bacteria. National Collections of Industrial and Marine Bacteria, Ltd., Aberdeen, Scotland.
22.	Neidle, E. L., and S. Kaplan. 1993. 5-aminolevulinic acid availability and control of spectral complex formation in HemA and HemT mutants of Rhodobacter sphaeroides. J. Bacteriol. 175:2304-2313[Abstract/Free Full Text].
23.	Neidle, E. L., and S. Kaplan. 1993. Expression of the Rhodobacter sphaeroides hemA and hemT genes, encoding two 5-aminolevulinic acid synthase isoenzymes. J. Bacteriol. 175:2292-2303[Abstract/Free Full Text].
23a.	Nereng, K. S., and S. Kaplan. Unpublished observations.
24.	Satoh, T., Y. Hoshino, and H. Kitamura. 1976. Rhodopseudomonas sphaeroides forma sp. denitrificans, a denitrifying strain as a subspecies of Rhodopseudomonas sphaeroides. Arch. Microbiol. 108:265-269[Medline].
25.	Sen, P., and N. Murai. 1991. Oligolabeling DNA probes to high specific activity with sequenase. Plant Mol. Biol. Rep. 9:127-130.
26.	Sistrom, W. R. 1962. The kinetics of the synthesis of photopigments in Rhodopseudomonas sphaeroides. J. Gen. Microbiol. 28:607-616[Medline].
27.	Suwanto, A., and S. Kaplan. 1989. Physical and genetic mapping of the Rhodobacter sphaeroides 2.4.1 genome: genome size, fragment identification, and gene localization. J. Bacteriol. 171:5840-5849[Abstract/Free Full Text].
28.	Suwanto, A., and S. Kaplan. 1989. Physical and genetic mapping of the Rhodobacter sphaeroides 2.4.1 genome: presence of two unique circular chromosomes. J. Bacteriol. 171:5850-5859[Abstract/Free Full Text].
29.	Suwanto, A., and S. Kaplan. 1992. A self-transmissible, narrow-host-range endogenous plasmid of Rhodobacter sphaeroides 2.4.1: physical structure, incompatibility determinants, origin of replication, and transfer functions. J. Bacteriol. 174:1124-1134[Abstract/Free Full Text].
30.	Tucker, W. T., and J. M. Pemberton. 1978. Viral R plasmid R6P: properties of the penicillinase plasmid prophage and the supercoiled, circular encapsidated genome. J. Bacteriol. 135:207-214[Abstract/Free Full Text].
31.	van Niel, C. B. 1944. The culture, general physiology, and classification of the non-sulfur purple and brown bacteria. Bacteriol. Rev. 8:1-118.
32.	Wall, J. D., P. F. Weaver, and H. Gest. 1975. Gene transfer agents, bacteriophages, and bacteriocins of Rhodopseudomonas capsulata. Arch. Microbiol. 105:217-224[Medline].
33.	Yoneda, M., H. Yaoi, M. Kida, and S. Matsuda. May 1972. U.S. patent 3,660,564.

This article has been cited by other articles:

• Choudhary, M., Zanhua, X., Fu, Y. X., Kaplan, S. (2007). Genome Analyses of Three Strains of Rhodobacter sphaeroides: Evidence of Rapid Evolution of Chromosome II. J. Bacteriol. 189: 1914-1921 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nereng, K. S. Articles by Kaplan, S. Search for Related Content PubMed PubMed Citation Articles by Nereng, K. S. Articles by Kaplan, S.