- Email: [email protected]
L12 genes from Brucella abortus
Gene. 140 (1994) 137-138 0 1994 Elsevier Science B.V. All rights reserved,
137
0378-l 119/94/%07.00
GENE 07703
Brief Notes
Sequences of the rpZJL operon containing the LlO and L7/L12 genes from Brucella abortus* (DNA sequencing;
ribosomal
proteins;
Sergio Costa Oliveira, Yingxun
Received by R.E. Yasbin: 30 August
bacterial
genes)
Zhu and Gary Splitter
1993; Revised/Accepted:
I4 October/l8
October
1993; Received at publishers:
15 November
1993
SUMMARY
The rplJL operon encodes the LlO and L7/L12 proteins, essential for ribosomal function and protein synthesis. In this study, we report the nucleotide sequence of the rplf and rplL genes from Brucella ahortus. The deduced aminoacid sequences show 37 and 67% identity to Esc~e~-ic~i~cofi LIO and L7/L12, respectively.
BrucelEu abortus is a facultative intracellular bacterium infecting animals and humans. This organism causes chronic infection and replicates within the cytoplasm of host mononuclear phagocytes. To understand the molecular mechanism used by B. nhortus to penetrate and resist destruction within the macrophages, we need to identify and study genes and their encoded products related to critical functions for the organism, like protein synthesis and bacterial replication. We report here the nt sequence of the LlO and L7/Ll2 genes that encode 50s r-proteins (Fig. 1). The LlO and L7/L12 proteins are essential in bacterial ribosomes for the proper function of factors like elongation factor-G (EF-G) and elongation factor-Tu (EF-Tu), involved in
Correspondence to: Gary A. Splitter, Department of Animal Health and Biomedical Sciences. University of Wisconsin, Madison, WI 53706. USA. Tel. ( t-608) 262-l 837; Fax (l-608) 262.7420; e-mail: GAS~ZEUS.AHABS.WISC.EDU *On request, the authors will supply detailed evidence for the conclusions reached in this Brief Note.
Abbreviations: aa, amino acid(s); B., Brucellu; BLAST, Basic Local Alignment Search Tool; bp. base pair(s); GCG, Genetic Computer Group (Madison, WI. USA); kb. kiJobase(s) or IOOObp; NCBI, National Center for Biotecllnoiogy information: nt, nucieotide(sj~ ohgo, oligodeoxyribonucleotide: psk-, pBluescript (Stratagene, La Jolla. CA, USA); r-, ribosomal.
SSDI
0378-I
119(93)E0699-E
protein synthesis (Liljas and Gudkov, 1987). In Esc~eric~li~~coii the genes encoding r-proteins LIO and L7jL12 are do-transcribed, and translation of both cistrons is regulated by binding of LlO or a complex of LlO and L7/L12 to a single target in the mRNA untranslated region that is located more than 100 nt upstream from the start codon (Climie and Friesen, 1988). Based upon sequence homology with the E. coti operon, we hypothesize that the same autogenous mechanism of translation regulation (feedback regulation) also takes place in the B. abortus rplJL operon. L7/L12 is the most studied among the r-proteins and exists as a dimer, present in the E. coii ribosome in four copies. The L7/L12 protein is attached to the 23s RNA via the LlO protein. L7L12 consists of a N-terminal domain which is necessary for binding to LlO and a C-terminal domain which is important in the binding of factors like EF-Tu and EF-G. L7/L12 is essential in the ribosome for maximal rate and low error frequency of protein synthesis (Rice and Steitz, 1989). LlO and L7/L12 r-cluster genes were isolated from B. abortus (strain 19) genomic DNA using a degenerate synthetic oligo to the 5’ end of the L7/L12 gene as a probe (Brooks-Worrel and Splitter, 1992). Among 38 B. ~~b~rt~s proteins analyzed by two-dimensional cellular immunoblotting, the L7/L12 was identified as the most
138
120 8 .
.
GTCGGACTGACTOTGAAGTTCGTCCGGCAAA OCCMCCCOGCAOOOCCMTCATCOTCCTGTCMCTOOACACA VGLTVKFVRQRQPGRANDGPVNWRlTVDRALIRLIVAnLN
240 48
GGcGcTTTcTcCoOTTcoOTcGTCGToocCC~TATACC~~~~CG~~~A~~~~~~GM~~G~A~~T~CGTT~G~~G~ GAFKLSGSVVVAllYTGLTVAQHSDLRSXI4RDAGGSVXVAK
360 86
AACCGCCTTGCCAAMTCGCTCTTCAGGGCACGGAATC
GGAAGGTATTGCTGACCTCTTTACGGGTCAGACGGTCGTTGCTTACGC~CGATCCGATCACCGCTCCG 480
NRLAKIALQGTl!SEGIADLFTGQTVVAYANDPITAPKVAV
128
GMTTC~CMDOCTMCCACM~GTTATTCTCOOTaCCDM
600 168
LFAKANDKLVILGGANGATTLKPRASSRFVRSRRSTNCAQ . AaC~TTOOTA~~CACACCCCOOCTCADCDTCT'PCGT s w L v l
720 172
.-. TTCTCGCTGTCMCAGTTCAMCCTTMTTATAGGMATAcAAMATGGcTGATC!EGcMAGATcGHADLAKIVLDLSALTVLEAA6LSKL CTcGiUGAGAAGTGGGGc GTTTCGGCTGClGCTCCGGTGC~TTGc~TGcC LLEKWGVSAAAPVAVAAAGGAAPAAAASSKTKFDVVLADG
CTTTCOOCCCTOACCGTTCTOOMOCCOCTOAOCTOTCCAATT
GGTGGCGCTGCCCCTGCTCCTGCCGCA' GAAGAM&cG~u~TT&ACGT~GTTC~~TGAC&
840 25 960 65
TGCGCGCACTCACCGGTCTCGGCCTCAAGGAAGCCAAGGACCTGGTCGMGGCGCTCCGAAGGC GGCGCTMCAAGA~MCGTGATCMGGMG TGTCAAGGMGGCGCCTCGAAGGAC! 1080 GANKINVIKl5VRALTGLGLKEAKDLVEGAPKAVKEGASKD 105 GM(3C~~MODCACA~~~T~~~C~T~T~T~~~CTATT ISAEKIXAQLlSAAGAKVLLK'
Fig. 1. Nucleotide each codon.
sequence
of the r-protein-encoding
1152 124
gene cluster from the rp/JL
operon.
The LIO begins at nt 97 and the L7/L12 gene begins at nt 766 (GenBank
immunodominant
protein
to freshly isolated
bovine
lym-
phocytes (Brooks-Worrel and Splitter, 1992). The B. ahortus genomic DNA was digested with Cl&, and the digested DNA was sized by agarose gel electrophoresis. Southern hybridization analysis, under low stringency conditions, was performed to identify the band containing both genes. The band was excised from the gel and the DNA purified using Geneclean (Bio 101, La Jolla, CA, USA). The isolated DNA fragment was then cloned into the Clal site of the psk- phagemid. Next, the vector carrying the LlO and L7/Ll2 genes was used to transform E. coli XLl-blue strain to amplify the genes of interest. Plasmid DNA was extracted using Magic Miniprep (Promega, Madison, WI, USA) and both genes were sequenced using a Sequenase version 2.0 kit (US Biochemical, Cleveland, OH, USA) by the Sanger dideoxy-mediated chain-termination method (Sambrook et al., 1989). To complete the entire operon sequence internal oligo primers were made at the Biotechnology Center of University of Wisconsin (Madison, WI, USA). The nt and deduced aa sequences of LlO and L7/L12 proteins are shown in the Fig. 1. The translation of the coding DNA was performed using a GCG sequence conversion program (Devereux et al., 1984). The LlO gene is 5 16 nt ( 172 aa) long and the L7/Ll2 is 372 nt ( 124 aa) long. Interestingly, not Met but Leu is the first aa of LlO. The same putative start codon is found in the L10 gene of citrus greening disease-associated bacterium that shares 50% identity with part of the deduced aa sequence of B. uhortus LlO gene (data not shown). The B. abortus
The predicted
accession
aa sequences
Nos. Ll9101
are given below the first nt of
and L23505).
LlO and L7/L12 share 37 and 67% identity with the equivalent deduced aa region of E. coli when the sequences of both genes were entered and searched in the GenBank using the BLAST network at the NCBI (Altschul et al., 1990). This work was supported by the College of Agricultural and Life Sciences and USDA grant 9237204-8114 and BARD I-1434-89. S.C.O. is a predoctoral Fellow supported by the Brazilian National Research Council (CNPq).
REFERENCES Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J.: Basic local alignment search tool. J. Mol. Biol. 215 (1990) 4033410. Brooks-Worrel, B. and Splitter, G.A.: Antigens of Bruce/la aborrus S19 immunodominant for bovine lymphocytes as identified by one- and two-dimensional cellular immunoblotting. Infect. Immun. 60 (1992) 245992464. Climie, S.C. and Friesen, J.D.: In riuu and in aitro structural analysis of the rp/JL mRNA leader of Escherichiu coli. Protection by bound LIO-L7/Ll2. J. Biol. Chem. 263 (1988) 15166-15175. Devereux, J., Haeberh, P. and Smithies, 0.: A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12 (1984) 387-395. Liljas, A. and Gudkov, A.T.: The structure and dynamics of ribosomal protein L12. Biochemie 69 (1987) 1043-1047. Rice, P.A. and Steitz, T.A.: Ribosomal protein L7/L12 has a helix-turnhelix motif similar to that found in DNA binding regulatory proteins. Nucleic Acids Res. 17 (1989) 3757 -3762. Sambrook, J., Fritsch, E.F. and Maniatis, T.: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 1989, pp. 13.6~13.10.
137
0378-l 119/94/%07.00
GENE 07703
Brief Notes
Sequences of the rpZJL operon containing the LlO and L7/L12 genes from Brucella abortus* (DNA sequencing;
ribosomal
proteins;
Sergio Costa Oliveira, Yingxun
Received by R.E. Yasbin: 30 August
bacterial
genes)
Zhu and Gary Splitter
1993; Revised/Accepted:
I4 October/l8
October
1993; Received at publishers:
15 November
1993
SUMMARY
The rplJL operon encodes the LlO and L7/L12 proteins, essential for ribosomal function and protein synthesis. In this study, we report the nucleotide sequence of the rplf and rplL genes from Brucella ahortus. The deduced aminoacid sequences show 37 and 67% identity to Esc~e~-ic~i~cofi LIO and L7/L12, respectively.
BrucelEu abortus is a facultative intracellular bacterium infecting animals and humans. This organism causes chronic infection and replicates within the cytoplasm of host mononuclear phagocytes. To understand the molecular mechanism used by B. nhortus to penetrate and resist destruction within the macrophages, we need to identify and study genes and their encoded products related to critical functions for the organism, like protein synthesis and bacterial replication. We report here the nt sequence of the LlO and L7/Ll2 genes that encode 50s r-proteins (Fig. 1). The LlO and L7/L12 proteins are essential in bacterial ribosomes for the proper function of factors like elongation factor-G (EF-G) and elongation factor-Tu (EF-Tu), involved in
Correspondence to: Gary A. Splitter, Department of Animal Health and Biomedical Sciences. University of Wisconsin, Madison, WI 53706. USA. Tel. ( t-608) 262-l 837; Fax (l-608) 262.7420; e-mail: GAS~ZEUS.AHABS.WISC.EDU *On request, the authors will supply detailed evidence for the conclusions reached in this Brief Note.
Abbreviations: aa, amino acid(s); B., Brucellu; BLAST, Basic Local Alignment Search Tool; bp. base pair(s); GCG, Genetic Computer Group (Madison, WI. USA); kb. kiJobase(s) or IOOObp; NCBI, National Center for Biotecllnoiogy information: nt, nucieotide(sj~ ohgo, oligodeoxyribonucleotide: psk-, pBluescript (Stratagene, La Jolla. CA, USA); r-, ribosomal.
SSDI
0378-I
119(93)E0699-E
protein synthesis (Liljas and Gudkov, 1987). In Esc~eric~li~~coii the genes encoding r-proteins LIO and L7jL12 are do-transcribed, and translation of both cistrons is regulated by binding of LlO or a complex of LlO and L7/L12 to a single target in the mRNA untranslated region that is located more than 100 nt upstream from the start codon (Climie and Friesen, 1988). Based upon sequence homology with the E. coti operon, we hypothesize that the same autogenous mechanism of translation regulation (feedback regulation) also takes place in the B. abortus rplJL operon. L7/L12 is the most studied among the r-proteins and exists as a dimer, present in the E. coii ribosome in four copies. The L7/L12 protein is attached to the 23s RNA via the LlO protein. L7L12 consists of a N-terminal domain which is necessary for binding to LlO and a C-terminal domain which is important in the binding of factors like EF-Tu and EF-G. L7/L12 is essential in the ribosome for maximal rate and low error frequency of protein synthesis (Rice and Steitz, 1989). LlO and L7/L12 r-cluster genes were isolated from B. abortus (strain 19) genomic DNA using a degenerate synthetic oligo to the 5’ end of the L7/L12 gene as a probe (Brooks-Worrel and Splitter, 1992). Among 38 B. ~~b~rt~s proteins analyzed by two-dimensional cellular immunoblotting, the L7/L12 was identified as the most
138
120 8 .
.
GTCGGACTGACTOTGAAGTTCGTCCGGCAAA OCCMCCCOGCAOOOCCMTCATCOTCCTGTCMCTOOACACA VGLTVKFVRQRQPGRANDGPVNWRlTVDRALIRLIVAnLN
240 48
GGcGcTTTcTcCoOTTcoOTcGTCGToocCC~TATACC~~~~CG~~~A~~~~~~GM~~G~A~~T~CGTT~G~~G~ GAFKLSGSVVVAllYTGLTVAQHSDLRSXI4RDAGGSVXVAK
360 86
AACCGCCTTGCCAAMTCGCTCTTCAGGGCACGGAATC
GGAAGGTATTGCTGACCTCTTTACGGGTCAGACGGTCGTTGCTTACGC~CGATCCGATCACCGCTCCG 480
NRLAKIALQGTl!SEGIADLFTGQTVVAYANDPITAPKVAV
128
GMTTC~CMDOCTMCCACM~GTTATTCTCOOTaCCDM
600 168
LFAKANDKLVILGGANGATTLKPRASSRFVRSRRSTNCAQ . AaC~TTOOTA~~CACACCCCOOCTCADCDTCT'PCGT s w L v l
720 172
.-. TTCTCGCTGTCMCAGTTCAMCCTTMTTATAGGMATAcAAMATGGcTGATC!EGcMAGATcGHADLAKIVLDLSALTVLEAA6LSKL CTcGiUGAGAAGTGGGGc GTTTCGGCTGClGCTCCGGTGC~TTGc~TGcC LLEKWGVSAAAPVAVAAAGGAAPAAAASSKTKFDVVLADG
CTTTCOOCCCTOACCGTTCTOOMOCCOCTOAOCTOTCCAATT
GGTGGCGCTGCCCCTGCTCCTGCCGCA' GAAGAM&cG~u~TT&ACGT~GTTC~~TGAC&
840 25 960 65
TGCGCGCACTCACCGGTCTCGGCCTCAAGGAAGCCAAGGACCTGGTCGMGGCGCTCCGAAGGC GGCGCTMCAAGA~MCGTGATCMGGMG TGTCAAGGMGGCGCCTCGAAGGAC! 1080 GANKINVIKl5VRALTGLGLKEAKDLVEGAPKAVKEGASKD 105 GM(3C~~MODCACA~~~T~~~C~T~T~T~~~CTATT ISAEKIXAQLlSAAGAKVLLK'
Fig. 1. Nucleotide each codon.
sequence
of the r-protein-encoding
1152 124
gene cluster from the rp/JL
operon.
The LIO begins at nt 97 and the L7/L12 gene begins at nt 766 (GenBank
immunodominant
protein
to freshly isolated
bovine
lym-
phocytes (Brooks-Worrel and Splitter, 1992). The B. ahortus genomic DNA was digested with Cl&, and the digested DNA was sized by agarose gel electrophoresis. Southern hybridization analysis, under low stringency conditions, was performed to identify the band containing both genes. The band was excised from the gel and the DNA purified using Geneclean (Bio 101, La Jolla, CA, USA). The isolated DNA fragment was then cloned into the Clal site of the psk- phagemid. Next, the vector carrying the LlO and L7/Ll2 genes was used to transform E. coli XLl-blue strain to amplify the genes of interest. Plasmid DNA was extracted using Magic Miniprep (Promega, Madison, WI, USA) and both genes were sequenced using a Sequenase version 2.0 kit (US Biochemical, Cleveland, OH, USA) by the Sanger dideoxy-mediated chain-termination method (Sambrook et al., 1989). To complete the entire operon sequence internal oligo primers were made at the Biotechnology Center of University of Wisconsin (Madison, WI, USA). The nt and deduced aa sequences of LlO and L7/L12 proteins are shown in the Fig. 1. The translation of the coding DNA was performed using a GCG sequence conversion program (Devereux et al., 1984). The LlO gene is 5 16 nt ( 172 aa) long and the L7/Ll2 is 372 nt ( 124 aa) long. Interestingly, not Met but Leu is the first aa of LlO. The same putative start codon is found in the L10 gene of citrus greening disease-associated bacterium that shares 50% identity with part of the deduced aa sequence of B. uhortus LlO gene (data not shown). The B. abortus
The predicted
accession
aa sequences
Nos. Ll9101
are given below the first nt of
and L23505).
LlO and L7/L12 share 37 and 67% identity with the equivalent deduced aa region of E. coli when the sequences of both genes were entered and searched in the GenBank using the BLAST network at the NCBI (Altschul et al., 1990). This work was supported by the College of Agricultural and Life Sciences and USDA grant 9237204-8114 and BARD I-1434-89. S.C.O. is a predoctoral Fellow supported by the Brazilian National Research Council (CNPq).
REFERENCES Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J.: Basic local alignment search tool. J. Mol. Biol. 215 (1990) 4033410. Brooks-Worrel, B. and Splitter, G.A.: Antigens of Bruce/la aborrus S19 immunodominant for bovine lymphocytes as identified by one- and two-dimensional cellular immunoblotting. Infect. Immun. 60 (1992) 245992464. Climie, S.C. and Friesen, J.D.: In riuu and in aitro structural analysis of the rp/JL mRNA leader of Escherichiu coli. Protection by bound LIO-L7/Ll2. J. Biol. Chem. 263 (1988) 15166-15175. Devereux, J., Haeberh, P. and Smithies, 0.: A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12 (1984) 387-395. Liljas, A. and Gudkov, A.T.: The structure and dynamics of ribosomal protein L12. Biochemie 69 (1987) 1043-1047. Rice, P.A. and Steitz, T.A.: Ribosomal protein L7/L12 has a helix-turnhelix motif similar to that found in DNA binding regulatory proteins. Nucleic Acids Res. 17 (1989) 3757 -3762. Sambrook, J., Fritsch, E.F. and Maniatis, T.: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 1989, pp. 13.6~13.10.