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Journal of Bacteriology, March 1999, p. 1684-1688, Vol. 181, No. 5
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
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.
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 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.
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Genomic Complexity among Strains of the Facultative
Photoheterotrophic Bacterium Rhodobacter
sphaeroides
ABSTRACT
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-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?
TABLE 1.
Bacterial strains and plasmids
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.
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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 2.4.1T.
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ACKNOWLEDGMENTS |
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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).
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FOOTNOTES |
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* 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.
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Journal of Bacteriology, March 1999, p. 1684-1688, Vol. 181, No. 5
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