Heat shock transcription of the groESL operon of Agrobacterium tumefaciens may involve a hairpin-loop structure

Segal, G. M.; Ron, Eliora Z.

doi:10.1128/jb.175.10.3083-3088.1993

Cited by 79 publications

(87 citation statements)

References 21 publications

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“…Our observation (Ogawa 1993) that multiple groEL copies seem to exist in other Rhizobium species and in Bradyrhizobium japonicum, but not in the closely related species A. tumefaciens (see also Fischer et al 1993;Segal and Ron 1993;Wallington and Lund 1994), may point to a role for the additional copies of groEL in the nodulation process. This is reinforced by the studies of Fischer et al (1993), who discovered five groEL genes in B. japonicum.…”

Section: Multiple Groel Locimentioning

confidence: 93%

The Rhizobium meliloti groELc locus is required for regulation of early nod genes by the transcription activator NodD.

Ogawa¹,

Long²

1995

Genes Dev.

107

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The molecular chaperones related to GroEL (hsp60, cpn60) interact with partially folded proteins and appear to assist them to attain active and correctly folded conformation. They are required for cell viability but are probably more important for some processes than for others. Through a random genetic search to find loci that are required for expression of the Rhizobium meliloti nod (nodulation) genes, we isolated a mutant (B4) defective in luteolin-dependent activation of nod gene expression, and found it carries a Tn5 insertion within a chromosomal groEL gene (groELc) located just downstream of a groESc gene. The groELc mutation affected activity of three related LysR-type activator proteins NodD1, NodD3, and SyrM; on plants, the mutants formed nodules late, and the nodules were Fix-. Hybridization and protein expression analysis show that a similar groESL locus (groESLa) maps to the Rml021 megaplasmid pSyma. Southern blot analysis revealed additional, but less closely related sequences hybridizing to groELc and groESc probes elsewhere in the R. meliloti genome. Clones of groESLc and groESLa can each restore robust phage ~ growth on an Escherichia coli groE mutant. Likewise each clone can complement all of the phenotypes observed for B4 mutants; thus, the two appear to be functionally equivalent if expression is controlled. We determined that groELc is required for normal DNA binding of the NodD target sequence in R. meliloti. GroEL coimmunopurifies with NodD1 from R. meliloti, which suggests a direct physical association between these proteins. GroEL is thus probably involved in the folding or assembly of transcriptionally active NodD.

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Section: Multiple Groel Locimentioning

confidence: 93%

The Rhizobium meliloti groELc locus is required for regulation of early nod genes by the transcription activator NodD.

Ogawa¹,

Long²

1995

Genes Dev.

107

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“…RNA preparation was performed as described elsewhere (42). The L. pneumophila JR32 strain was grown on ABCYE plates for 48 h. The bacteria were scraped off the plate and suspended in AYE broth, and the bacterial OD 600 was calibrated to 0.1 in AYE.…”

Section: Methodsmentioning

confidence: 99%

Analysis of DNA Regulatory Elements Required for Expression of theLegionella pneumophilaicmanddotVirulence Genes

2002

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To investigate the regulation of the Legionella pneumophila icm and dot genes required for intracellular growth, a series of nine icm::lacZ fusions were constructed. These icm::lacZ fusions were found to have different levels of expression in L. pneumophila, and five of them were more highly expressed at stationary phase than at exponential phase. When the expression of these fusions in Escherichia coli was tested, all of them were found to be expressed but three of them had dramatic changes in their levels of expression in comparison to those in L. pneumophila. Site-directed and PCR random mutagenesis with these icm::lacZ fusions was used to identify DNA regulatory elements of icm genes. Four icm genes (icmT, icmP, icmQ, and icmM) that had low levels of expression in L. pneumophila were found to contain a 6-bp sequence (TATACT) essential for their expression. This sequence was shown by primer extension to serve as their ؊10 promoter elements. A similar sequence, which constitutes the ؊10 promoter elements of the icmV, icmW, and icmR genes which had high levels of expression in L. pneumophila, was also identified. In addition, regulatory elements that probably serve as binding sites for transcription regulators were found in these genes. Altogether, 12 regulatory elements, 7 of which constitute the ؊10 promoter elements of the icm genes, were found. Even though all the icm and dot genes are part of one system required for L. pneumophila intracellular growth and even though their promoters are probably recognized by the vegetative sigma factor, it seems that they are subjected to different regulation mediated by several regulatory factors.Legionella pneumophila, the causative agent of Legionnaires' disease, is a facultative intracellular pathogen. L. pneumophila is able to infect, multiply within, and kill human macrophages, as well as free-living amoebae (28, 38). The bacteria are taken up by regular phagocytosis or by a special mechanism termed "coiling" phagocytosis (9, 26). They are then found within a specialized phagosome that does not fuse with lysosomes or acidify (9,24,27). The specialized phagosome undergoes several recruitment events, which include association with smooth vesicles, mitochondria, and the rough endoplasmic reticulum (1,25,49); the bacteria multiply within the specialized phagosome until the cell eventually lyses, releasing bacteria that can start new rounds of infection (28,38).Two regions of genes required for human macrophage killing and intracellular multiplication have been discovered in L. pneumophila (reviewed in references 44 and 51). Region I contains 7 genes (icmV, -W, and -X and dotA, -B, -C, and -D) (8, 10, 32, 50), and region II contains 17 genes (icmT, -S , -R, -Q, -P, -O, -N, -M, -L, -K, -E, -G, -C, -D, -J, -B, and -F) (3,35,41,43,50). Complementation analysis of these genes indicated that they are probably organized as nine transcriptional units (icmTS, icmR, icmQ, icmPO, icmMLKEGCD, icmJB, icmFtphA, icmWX, and icmV-dotA) (8,10,35,41,43,50). Most of these genes were also...

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“…In these organisms, only a single heat shock groESL operon was cloned after screening with E. coli groESL probes. Our initial library screen with the B. japonicum groESL 2 probe also identified (35) and Clostridium acetobutylicum (28), transcription begins at the 5Ј end of the conserved hairpin loop. The putative Ϫ35 and Ϫ10 sequences present upstream of the R. sphaeroides groESL genes are conserved in 5Ј sequences of heat shock genes from several bacteria.…”

Section: Resultsmentioning

confidence: 99%

“…Within this subdivision are B. japonicum and R. meliloti, both of which possess multiple groESL operons. Other groESL genes from this subdivision have been cloned from Brucella abortus (16) and Agrobacterium tumefaciens (35). In these organisms, only a single heat shock groESL operon was cloned after screening with E. coli groESL probes.…”

Section: Resultsmentioning

confidence: 99%

Cloning and characterization of two groESL operons of Rhodobacter sphaeroides: transcriptional regulation of the heat-induced groESL operon

1997

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The nonsulfur purple bacterium Rhodobacter sphaeroides was found to contain two groESL operons. The groESL 1 heat shock operon was cloned from a genomic library, and a 2.8-kb DNA fragment was sequenced and found to contain the groES and groEL genes. The deduced amino acid sequences of GroEL 1 (cpn60) and GroES 1 (cpn10) were in agreement with N-terminal sequences previously obtained for the isolated proteins (K. C. Terlesky and F. R. Tabita, Biochemistry 30:8181-8186, 1991). These sequences show a high degree of similarity to groESL genes isolated from other bacteria. Northern analysis indicated that the groESL 1 genes were expressed as part of a 2.2-kb polycistronic transcript that is induced 13-fold after heat shock. Transcript size was not affected by heat shock; however, the amount of transcript was induced to its greatest extent 15 to 30 min after a 40؇C heat shock, from an initial temperature of 28؇C, and remained elevated up to 120 min. The R. sphaeroides groESL 1 operon contains a putative hairpin loop at the start of the transcript that is present in other bacterial heat shock genes. Primer extension of the message showed that the transcription start site is at the start of this conserved hairpin loop. In this region were also found putative ؊35 and ؊10 sequences that are conserved upstream from other bacterial heat shock genes. Transcription of the groESL 1 genes was unexpectedly low under photoautotrophic growth conditions. Thus far, it has not been possible to construct a groESL 1 deletion strain, perhaps indicating that these genes are essential for growth. A second operon (groESL 2 ) was also cloned from R. sphaeroides, using a groEL 1 gene fragment as a probe; however, no transcript was observed for this operon under several different growth conditions. A groESL 2 deletion strain was constructed, but there was no detectable change in the phenotype of this strain compared to the parental strain.

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Heat shock transcription of the groESL operon of Agrobacterium tumefaciens may involve a hairpin-loop structure

Cited by 79 publications

References 21 publications

The Rhizobium meliloti groELc locus is required for regulation of early nod genes by the transcription activator NodD.

The Rhizobium meliloti groELc locus is required for regulation of early nod genes by the transcription activator NodD.

Analysis of DNA Regulatory Elements Required for Expression of theLegionella pneumophilaicmanddotVirulence Genes

Cloning and characterization of two groESL operons of Rhodobacter sphaeroides: transcriptional regulation of the heat-induced groESL operon

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