Identification and characterization of the ilvR gene encoding a LysR-type regulator of Caulobacter crescentus

Malakooti, Jaleh; Ely, Bert

doi:10.1128/jb.176.5.1275-1281.1994

Cited by 11 publications

(7 citation statements)

References 36 publications

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“…Plasmid DNA extraction, DNA digestion, ligation, and transformation of E. coli host cells were carried out as described by Sambrook et al (27). Electroporation was performed as described previously (18). All restriction and modifying enzymes were obtained from New England Biolabs Inc. (Beverly, Mass.…”

Section: Methodsmentioning

confidence: 99%

Principal sigma subunit of the Caulobacter crescentus RNA polymerase

Malakooti

Ely

1995

J Bacteriol

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We have identified the gene encoding the Caulobacter crescentus principal sigma subunit, RpoD. The rpoD gene codes for a polypeptide of 653 amino acids with a predicted molecular mass of 72,623 Da ( 73 ). The C. crescentus sigma subunit has extensive amino acid sequence homology with the principal sigma factors of a number of divergent procaryotes. In particular, the segments designated region 2 that are involved in core polymerase binding and promoter recognition were identical among these bacteria despite the fact that the ؊10 region recognized by the C. crescentus 73 differs significantly from that of the other bacteria. Thus, it appears that additional sigma factor regions must be involved in ؊10 region recognition. This conclusion was strengthened by a heterologous complementation assay in which C. crescentus 73 was capable of complementing the Escherichia coli rpoD285 temperature-sensitive mutant. Furthermore, C. crescentus 73 conferred new specificity on the E. coli RNA polymerase, allowing the expression of C. crescentus promoters in E. coli. Thus, the C. crescentus 73 appears to have a broader specificity than does the 70 of the enteric bacteria.Caulobacter crescentus is a dimorphic, gram-negative bacterium with a well-defined cell cycle. During the processes of cell differentiation and cell division, a number of morphological and biochemical changes result in the conversion of a stalked cell into two dissimilar progeny cells. One of the progeny is the same as the parent, a sessile stalked cell which can immediately initiate DNA replication and the process of cell division. The other is a motile swarmer cell which is not competent for DNA replication. Before the swarmer cell can commence DNA replication and the subsequent cell differentiation events, it must lose its flagellum and generate a stalk in the place previously occupied by the flagellum. Once the stalk is formed, the cell initiates DNA replication and proceeds through the cell cycle (for reviews, see references 31 and 22). The cell cycle events resulting in and from cell differentiation are dependent on de novo RNA synthesis (21), suggesting that the regulation of gene expression in this system may be controlled at the level of transcription. The expression of a number of flagellar genes is regulated by an alternative form of the sigma subunit (5, 20) and additional trans-acting regulatory factors (23,25). Thus, modification of the transcriptional apparatus seems to be one way by which differential gene expression occurs.The purified RNA polymerase of C. crescentus is composed of subunits analogous to those of other eubacterial RNA polymerases (3). Furthermore, Amemiya and Shapiro (1) demonstrated that the C. crescentus RNA polymerase recognizes the same promoter and terminator signals as the Escherichia coli enzyme. However, C. crescentus promoters are not recognized by E. coli RNA polymerase (1,35,40). Furthermore, we have shown that the C. crescentus promoter sequences are different from those of the canonical promoter sequence recognized b...

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Section: Methodsmentioning

confidence: 99%

Principal sigma subunit of the Caulobacter crescentus RNA polymerase

Malakooti

Ely

1995

J Bacteriol

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“…The ilvR and ilvD genes of C. crescentus are divergently transcribed from a common regulatory region, and the ilvR gene product exhibits homology to the LysR family of transcriptional activators (15). The members of this family of transcriptional regulators often are activators of upstream genes that are divergently transcribed (12).…”

Section: Determination Of the 5 Terminus Of The Ilvd And The Ilvr Tramentioning

confidence: 99%

“…The members of this family of transcriptional regulators often are activators of upstream genes that are divergently transcribed (12). Similarly, the IlvR protein was shown to be a transcriptional activator of the upstream and divergently transcribed ilvD gene (15). To map the transcription initiation site of the ilvD transcript, a NarIXhoI fragment containing the promoter regions for both the ilvD and ilvR genes was labeled at the NarI 5Ј end and hybridized to total cellular RNA.…”

Section: Determination Of the 5 Terminus Of The Ilvd And The Ilvr Tramentioning

confidence: 99%

“…It is not known whether the smaller protected fragment represents an independent transcript or a degradation product of the larger transcript. The assignment of the ATG start codon to this position was based on the lack of any other potential start codon and the amino acid sequence homology of the ilvR protein with other LysR-type proteins (15). A summary of the promoter regions for ilvD and ilvR is shown in Fig.…”

Section: Vol 177 1995 C Crescentus Biosynthetic Gene Promoter 4373mentioning

confidence: 99%

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A consensus promoter sequence for Caulobacter crescentus genes involved in biosynthetic and housekeeping functions

1995

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Caulobacter crescentus differentiates prior to each cell division to form two different daughter cells: a monoflagellated swarmer cell and a nonmotile stalked cell. Thus, one might expect that developmentally expressed genes would be regulated by mechanisms different from those used to regulate the expression of the biosynthetic genes. To determine a consensus promoter sequence for genes involved in biosynthetic or housekeeping functions, DNA fragments containing the regulatory regions of the ilvD, ilvR, cysC, pleC, and fdxA genes were cloned. S1 nuclease protection mapping and primer extension techniques were used to identify the transcription initiation sites. Comparison of the regulatory regions of these genes with those of the published sequences of the ilvBN, rrnA, trpFBA, dnaA, dnaK, hemE, and rsaA genes has resulted in the identification of a putative promoter consensus sequence. The ؊35 region contains the sequence TTGACGS, which is similar to the Escherichia coli ؊35 region, while the ؊10 region, GCTANAWC, has a more balanced GC content than the corresponding region in E. coli. Oligonucleotide-directed site-specific mutagenesis of both the ilvBN and pleC promoters indicates that mutations that make a promoter more like the consensus result in increased promoter activity, while mutations decreasing similarity to the consensus result in decreased promoter activity.Caulobacter crescentus is a gram-negative bacterium that differentiates prior to each cell division to generate two dissimilar progeny cells. The new cells differ from one another morphologically and developmentally. Much of what is known about gene regulation in C. crescentus has resulted from studies of periodically expressed genes, especially the genes involved in flagellar biogenesis and function (21, 27). The 5Ј regulatory regions of some of the flagellar genes have been shown to contain a set of activated promoters that are transcribed by a 54 RNA polymerase holoenzyme (3,19). These promoters contain the consensus sequences recognized by the enteric 54 at positions Ϫ12 and Ϫ24 (4,19,22). Other fla genes may be regulated by a second alternative sigma factor (5,30,35).Very little information is available on the expression of genes that are expected to show no cell cycle-dependent regulation. For instance, biosynthetic genes are likely to be expressed throughout the cell cycle to fulfill the nutritional requirements of the bacterium. To understand the regulatory mechanisms involved in the expression of the biosynthetic and housekeeping genes, we investigated the cis-acting regulatory elements involved in the expression of a number of such genes. Analysis of the sequences within the 5Ј regulatory regions led us to propose a consensus promoter sequence for biosynthetic and housekeeping genes. Site-specific mutagenesis of the ilvBN and the pleC promoter regions was used to evaluate the role of the nucleotides composing the proposed consensus. MATERIALS AND METHODSBacterial growth. C. crescentus strains were grown in PYE medium (13), and Escheric...

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“…The IlvR protein was the only LTTR studied so far in C. crescentus. IlvR positively regulates the expression of the ilvD gene, which is involved in isoleucine/ valine biosynthesis, and negatively autoregulates its own expression; however, the coinducer necessary for its activation was not identified (18). Members of this family usually have diverse cellular functions and bind DNA through a conserved N-terminal domain in response to the binding of a coinducer, usually a small molecule.…”

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CztR, a LysR-Type Transcriptional Regulator Involved in Zinc Homeostasis and Oxidative Stress Defense in Caulobacter crescentus

et al. 2010

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Caulobacter crescentus is a free-living alphaproteobacterium that has 11 predicted LysR-type transcriptional regulators (LTTRs). Previously, a C. crescentus mutant strain with a mini-Tn5lacZ transposon inserted into a gene encoding an LTTR was isolated; this mutant was sensitive to cadmium. In this work, a mutant strain with a deletion was obtained, and the role of this LTTR (called CztR here) was evaluated. The transcriptional start site of this gene was determined by primer extension analysis, and its promoter was cloned in front of a lacZ reporter gene. ␤-Galactosidase activity assays, performed with the wild-type and mutant strains, indicated that this gene is 2-fold induced when cells enter stationary phase and that it is negatively autoregulated. Moreover, this regulator is essential for the expression of the divergent cztA gene at stationary phase, in minimal medium, and in response to zinc depletion. This gene encodes a hypothetical protein containing 10 predicted transmembrane segments, and its expression pattern suggests that it encodes a putative zinc transporter. The cztR strain was also shown to be sensitive to superoxide (generated by paraquat) and to hydrogen peroxide but not to tert-butyl hydroperoxide. The expression of katG and ahpC, but not that of the superoxide dismutase genes, was increased in the cztR mutant. A model is proposed to explain how CztR binding to the divergent regulatory regions could activate cztA expression and repress its own transcription.

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Identification and characterization of the ilvR gene encoding a LysR-type regulator of Caulobacter crescentus

Cited by 11 publications

References 36 publications

Principal sigma subunit of the Caulobacter crescentus RNA polymerase

Principal sigma subunit of the Caulobacter crescentus RNA polymerase

A consensus promoter sequence for Caulobacter crescentus genes involved in biosynthetic and housekeeping functions

CztR, a LysR-Type Transcriptional Regulator Involved in Zinc Homeostasis and Oxidative Stress Defense in Caulobacter crescentus

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