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Papers by Rachel Muir

Evolution of Microbial Pathogens, 2006

EVOLUTION OF PATHOGENS IN SOIL Rachel Muir and Man-Wah Tan 8 The soil is a particularly complex e... more EVOLUTION OF PATHOGENS IN SOIL Rachel Muir and Man-Wah Tan 8 The soil is a particularly complex environment and is one of the most dynamic sites of biologi-cal interactions in nature. It is not merely a static physicochemical matrix, but a biological system in a ...

Molecular Microbiology, 2002

Mutations in FlbD that relieve the dependency on flagellum assembly alter the temporal and spatia... more Mutations in FlbD that relieve the dependency on flagellum assembly alter the temporal and spatial pattern of developmental transcription in Caulobacter crescentus expression and their capacity to reinitiate chromosomal DNA replication (reviewed in Brun et al., 1994; Gober and Marques, 1995; Wu and Newton, 1997; Gober and England, 2000). For example, the newly formed stalked cell initiates DNA replication almost immediately after cell division, whereas replication is repressed for a period of time in the swarmer cell. Following this period of repression, the flagellum is shed, a stalk is synthesized in its place and DNA replication initiates (Fig. 1). This programme of cellular differentiation is directed, in part, by both cell cycle and spatial transcription which is regulated by members of the large family of bacterial two-component regulatory systems. The basic regulatory paradigm common to these signal transduction systems consists of a stimulatory cue, often environmental, which is sensed by and, in turn, activates autophosphorylation of a sensor histidine kinase (reviewed in Parkinson and Kofoid, 1992). The phosphate from this kinase is then transferred to a conserved receiver domain of a response regulator protein that, very often, is a transcription factor. One hallmark of the C. crescentus programme of cellular differentiation is the temporal, and spatial, biogenesis of a single polar flagellum at the pole opposite the stalk (Fig. 1). The synthesis of this flagellum is regulated by two distinct global-response regulator transcription factors, CtrA and FlbD, that function at specific times and locations in the pre-divisional cell (reviewed in Brun et al., 1994; Gober and Marques, 1995; Wu and Newton, 1997; Gober and England, 2000). The biogenesis of the polar flagellum requires at least 50 gene products and is regulated by a complex transacting hierarchy that is influenced both by progression of the cell cycle and flagellum assembly. The earliest synthesized flagellar components consist of those that encode the MS-ring (fliF), the flagellar switch and components of the flagellum-specific secretory system. These early, class II genes share a conserved promoter sequence that contains a binding site for the transcription factor, CtrA (Quon et al., 1996; Domian et al., 1997; Reisenauer et al., 1999). CtrA is activated by a cell cycle cue, which is presumably linked to the initiation of DNA replication. Following the expression and assembly of the early, class II-encoded flagellar structure, the genes encoding Molecular Microbiology (2002) 43(3), 597-615

Molecular Microbiology, 2001

The first flagellar assembly checkpoint of Caulobacter crescentus couples assembly of the early c... more The first flagellar assembly checkpoint of Caulobacter crescentus couples assembly of the early class II components of the basal body complex to the expression of class III and IV genes, which encode extracytoplasmic structures of the flagellum. The transcription of class III/IV flagellar genes is activated by the response regulator factor, FlbD. Gain of function mutations in flbD, termed bfa, can bypass the transcriptional requirement for the assembly of class II flagellar structures. Here we show that the class II flagellar gene fliX encodes a transacting factor that couples flagellar assembly to FlbD-dependent transcription. We show that the overexpression of fliX can suppress class III/IV gene expression in both wild-type and flbD-bfa cells. Introduction of a bfa allele of flbD into cells possessing a deletion in fliX restores motility indicating that FliX is not a structural component of the flagellum, but rather a transacting factor. Furthermore, extragenic motile suppressors which arise in DfliX cells map to the flbD locus. These results indicate that FlbD functions downstream of FliX in activating class III/IV transcription. b-Lactamase fusions to FliX and analysis of cellular fractions demonstrate that FliX is a cytosolic protein that demonstrates some peripheral association with the cytoplasmic membrane. In addition, we have isolated a mutant allele of fliX that exhibits a bfa-like phenotype, restoring flbD-dependent class III/IV transcription in strains that contain mutations in class II flagellar structural genes. Taken together, these results indicated both a positive and negative regulatory function for FliX in coupling the assembly of class II basal body components to gene expression.

Molecular Microbiology, 2001

Biogenesis of the single polar flagellum of Caulobacter crescentus is regulated by a complex inte... more Biogenesis of the single polar flagellum of Caulobacter crescentus is regulated by a complex interplay of cell cycle events and the progression of flagellum assembly. The expression of class III/IV flagellar genes requires the assembly of an early flagellar basal body structure, encoded by class II genes, and is activated by the transcription factor FlbD. Previous experiments indicated that the class II flagellar gene, flbE, encoded a transacting factor that was required for FlbD activity. Here, using mutant alleles of flbE we have determined that FlbE is either a structural component of the flagellum or is required for flagellar assembly and does not, as originally proposed, function as a transacting factor. We also demonstrate that two deleted derivatives of flbE have a dominant negative effect on the transcriptional activation of class III/IV flagellar genes that can be relieved by a gain-of-function mutation in flbD called bfa. This same mutation in flbD has been shown to restore class III/IV transcription in the absence of early class II flagellar assembly. These deleted mutants of flbE also exhibited a filamentous cell phenotype that was indistinguishable from that previously observed in class II flagellar mutants. Introduction of a flbD-bfa mutation into these cells expressing the deleted alleles of flbE, as well as several class II mutant strains, restored normal cell division and FtsZ localization. These results suggest that class III/IV transcription and a step in cell division are coupled to flagellar assembly by the same genetic pathway.

Molecular Microbiology, 2004

The temporal and spatial transcription of late flagellar genes in Caulobacter crescentus is regul... more The temporal and spatial transcription of late flagellar genes in Caulobacter crescentus is regulated by the s s s s 54 transcriptional activator, FlbD. One requirement for FlbD activity is the assembly of a structure encoded by early, class II flagellar genes. In this report, we show that the transacting factor FliX predominantly functions as a negative regulator of FlbD activity in the absence of the class II-encoded flagellar structure. In contrast, a mutant FliX that bypasses the transcriptional requirement for early flagellar assembly is incapable of repressing FlbD in a class II flagellar mutant. Expression of this mutant allele, fliX1 , does not alter the temporal pattern of FlbD-dependent transcription. Remarkably, this mutation confers the correct cell cycle timing of hook operon transcription in a strain that cannot assemble the flagellum, indicating that the progression of flagellar assembly is a minor influence on temporal gene expression. Using a two-hybrid assay, we present evidence that FliX regulates FlbD through a direct interaction, a novel mechanism for this class of s s s s 54 transcriptional activator. Furthermore, increasing the cellular levels of FliX results in an increase in the concentration of FlbD, and a corresponding increase in FlbD-activated transcription, suggesting that FliX and FlbD form a stable complex in Caulobacter. FliX and FlbD homologues are present in several polar-flagellated bacteria, indicating that these proteins constitute an evolutionarily conserved regulatory pair in organisms where flagellar biogenesis is likely to be under control of the cell division cycle.

Journal of Bacteriology, 2005

In the Caulobacter crescentus predivisional cell, class III and IV flagellar genes, encoding the ... more In the Caulobacter crescentus predivisional cell, class III and IV flagellar genes, encoding the extracytoplasmic components of the flagellum, are transcribed in the nascent swarmer compartment. This asymmetric expression pattern is attributable to the compartmentalized activity of the σ54-dependent transcriptional activator FlbD. Additionally, these temporally transcribed flagellar promoters possess a consensus sequence for the DNA-binding protein integration host factor (IHF), located between the upstream FlbD binding site and the promoter sequences. Here, we deleted the C. crescentus gene encoding the β-subunit of the IHF, ihfB (himD), and examined the effect on flagellar gene expression. The ΔihfB strain exhibited a mild defect in cell morphology and impaired motility. Using flagellar promoter reporter fusions, we observed that expression levels of a subset of class III flagellar promoters were decreased by the loss of IHF. However, one of these promoters, fliK-lacZ, exhibited a...

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, 2007

A yellow-pigmented, Gram-positive, aerobic, non-motile, non-spore-forming, irregular rod-shaped b... more A yellow-pigmented, Gram-positive, aerobic, non-motile, non-spore-forming, irregular rod-shaped bacterium (strain TAN 31504T) was isolated from the bacteriophagous nematode Caenorhabditis elegans. Based on 16S rRNA gene sequence similarity, DNA G+C content of 69.5 mol%, 2,4-diaminobutyric acid in the cell-wall peptidoglycan, major menaquinone MK-11, abundance of anteiso- and iso-fatty acids, polar lipids diphosphatidylglycerol and phosphatidylglycerol and a number of shared biochemical characteristics, strain TAN 31504T was placed in the genus Leucobacter. DNA–DNA hybridization comparisons demonstrated a 91 % DNA–DNA relatedness between strain TAN 31504T and Leucobacter chromiireducens LMG 22506T indicating that these two strains belong to the same species, when the recommended threshold value of 70 % DNA–DNA relatedness for the definition of a bacterial species by the ad hoc committee on reconciliation of approaches to bacterial systematics is considered. Based on distinct differen...

Applied and Environmental Microbiology, 2008

We describe the pathogenic interaction between a newly described gram-positive bacterium, Leucoba... more We describe the pathogenic interaction between a newly described gram-positive bacterium, Leucobacter chromiireducens subsp. solipictus strain TAN 31504, and the nematode Caenorhabditis elegans . TAN 31504 pathogenesis on C. elegans is exerted primarily through infection of the adult nematode uterus. TAN 31504 enters the uterus through the external vulval opening, and the ensuing uterine infection is strongly correlated with a significant reduction in host life span. Young worms can feed and develop on TAN 31504, but not preferably over the standard food source. C. elegans worms reared on TAN 31504 as the sole food source develop into thin adults with little intestinal fat stores, produce few progeny, and subsequently cannot persist on the pathogenic food source. Within 12 h of exposure, adult worms challenged with TAN 31504 alter the expression of a number of C. elegans innate immunity-related genes, including nlp-29 , which encodes a neuropeptide-like protein. C. elegans worms exp...

Evolution of microbial …, 2006

EVOLUTION OF PATHOGENS IN SOIL Rachel Muir and Man-Wah Tan 8 The soil is a particularly complex e... more EVOLUTION OF PATHOGENS IN SOIL Rachel Muir and Man-Wah Tan 8 The soil is a particularly complex environment and is one of the most dynamic sites of biologi-cal interactions in nature. It is not merely a static physicochemical matrix, but a biological system in a ...

International journal of systematic and …, 2007

A yellow-pigmented, Gram-positive, aerobic, non-motile, non-spore-forming, irregular rod-shaped b... more A yellow-pigmented, Gram-positive, aerobic, non-motile, non-spore-forming, irregular rod-shaped bacterium (strain TAN 31504 T) was isolated from the bacteriophagous nematode Caenorhabditis elegans. Based on 16S rRNA gene sequence similarity, DNA G+C content of 69.5 mol%, 2,4-diaminobutyric acid in the cell-wall peptidoglycan, major menaquinone MK-11, abundance of anteiso-and iso-fatty acids, polar lipids diphosphatidylglycerol and phosphatidylglycerol and a number of shared biochemical characteristics, strain TAN 31504 T was placed in the genus Leucobacter. DNA-DNA hybridization comparisons demonstrated a 91 % DNA-DNA relatedness between strain TAN 31504 T and Leucobacter chromiireducens LMG 22506 T indicating that these two strains belong to the same species, when the recommended threshold value of 70 % DNA-DNA relatedness for the definition of a bacterial species by the ad hoc committee on reconciliation of approaches to bacterial systematics is considered. Based on distinct differences in morphology, physiology, chemotaxonomic markers and various biochemical characteristics, it is proposed to split the species L. chromiireducens into two novel subspecies, Leucobacter chromiireducens subsp. chromiireducens subsp. nov. (type strain L-1 T 5CIP 108389 T 5LMG 22506 T) and Leucobacter chromiireducens subsp. solipictus subsp. nov. (type strain TAN 31504 T 5DSM 18340 T 5ATCC BAA-1336 T).

Applied and environmental microbiology, 2008

We describe the pathogenic interaction between a newly described gram-positive bacterium, Leucoba... more We describe the pathogenic interaction between a newly described gram-positive bacterium, Leucobacter chromiireducens subsp. solipictus strain TAN 31504, and the nematode Caenorhabditis elegans. TAN 31504 pathogenesis on C. elegans is ...

Molecular microbiology, 2004

The temporal and spatial transcription of late flagellar genes in Caulobacter crescentus is regul... more The temporal and spatial transcription of late flagellar genes in Caulobacter crescentus is regulated by the s s s s 54 transcriptional activator, FlbD. One requirement for FlbD activity is the assembly of a structure encoded by early, class II flagellar genes. In this report, we show that the transacting factor FliX predominantly functions as a negative regulator of FlbD activity in the absence of the class II-encoded flagellar structure. In contrast, a mutant FliX that bypasses the transcriptional requirement for early flagellar assembly is incapable of repressing FlbD in a class II flagellar mutant. Expression of this mutant allele, fliX1 , does not alter the temporal pattern of FlbD-dependent transcription. Remarkably, this mutation confers the correct cell cycle timing of hook operon transcription in a strain that cannot assemble the flagellum, indicating that the progression of flagellar assembly is a minor influence on temporal gene expression. Using a two-hybrid assay, we present evidence that FliX regulates FlbD through a direct interaction, a novel mechanism for this class of s s s s 54 transcriptional activator. Furthermore, increasing the cellular levels of FliX results in an increase in the concentration of FlbD, and a corresponding increase in FlbD-activated transcription, suggesting that FliX and FlbD form a stable complex in Caulobacter. FliX and FlbD homologues are present in several polar-flagellated bacteria, indicating that these proteins constitute an evolutionarily conserved regulatory pair in organisms where flagellar biogenesis is likely to be under control of the cell division cycle.

Molecular microbiology, 2002

Mutations in FlbD that relieve the dependency on flagellum assembly alter the temporal and spatia... more Mutations in FlbD that relieve the dependency on flagellum assembly alter the temporal and spatial pattern of developmental transcription in Caulobacter crescentus expression and their capacity to reinitiate chromosomal DNA replication (reviewed in Brun et al., 1994; Gober and Marques, 1995; Wu and Newton, 1997; Gober and England, 2000). For example, the newly formed stalked cell initiates DNA replication almost immediately after cell division, whereas replication is repressed for a period of time in the swarmer cell. Following this period of repression, the flagellum is shed, a stalk is synthesized in its place and DNA replication initiates (Fig. 1). This programme of cellular differentiation is directed, in part, by both cell cycle and spatial transcription which is regulated by members of the large family of bacterial two-component regulatory systems. The basic regulatory paradigm common to these signal transduction systems consists of a stimulatory cue, often environmental, which is sensed by and, in turn, activates autophosphorylation of a sensor histidine kinase (reviewed in Parkinson and Kofoid, 1992). The phosphate from this kinase is then transferred to a conserved receiver domain of a response regulator protein that, very often, is a transcription factor. One hallmark of the C. crescentus programme of cellular differentiation is the temporal, and spatial, biogenesis of a single polar flagellum at the pole opposite the stalk (Fig. 1). The synthesis of this flagellum is regulated by two distinct global-response regulator transcription factors, CtrA and FlbD, that function at specific times and locations in the pre-divisional cell (reviewed in Brun et al., 1994; Gober and Marques, 1995; Wu and Newton, 1997; Gober and England, 2000). The biogenesis of the polar flagellum requires at least 50 gene products and is regulated by a complex transacting hierarchy that is influenced both by progression of the cell cycle and flagellum assembly. The earliest synthesized flagellar components consist of those that encode the MS-ring (fliF), the flagellar switch and components of the flagellum-specific secretory system. These early, class II genes share a conserved promoter sequence that contains a binding site for the transcription factor, CtrA (Quon et al., 1996; Domian et al., 1997; Reisenauer et al., 1999). CtrA is activated by a cell cycle cue, which is presumably linked to the initiation of DNA replication. Following the expression and assembly of the early, class II-encoded flagellar structure, the genes encoding Molecular Microbiology (2002) 43(3), 597-615

Molecular microbiology, 2001

The first flagellar assembly checkpoint of Caulobacter crescentus couples assembly of the early c... more The first flagellar assembly checkpoint of Caulobacter crescentus couples assembly of the early class II components of the basal body complex to the expression of class III and IV genes, which encode extracytoplasmic structures of the flagellum. The transcription of class III/IV flagellar genes is activated by the response regulator factor, FlbD. Gain of function mutations in flbD, termed bfa, can bypass the transcriptional requirement for the assembly of class II flagellar structures. Here we show that the class II flagellar gene fliX encodes a transacting factor that couples flagellar assembly to FlbD-dependent transcription. We show that the overexpression of fliX can suppress class III/IV gene expression in both wild-type and flbD-bfa cells. Introduction of a bfa allele of flbD into cells possessing a deletion in fliX restores motility indicating that FliX is not a structural component of the flagellum, but rather a transacting factor. Furthermore, extragenic motile suppressors which arise in DfliX cells map to the flbD locus. These results indicate that FlbD functions downstream of FliX in activating class III/IV transcription. b-Lactamase fusions to FliX and analysis of cellular fractions demonstrate that FliX is a cytosolic protein that demonstrates some peripheral association with the cytoplasmic membrane. In addition, we have isolated a mutant allele of fliX that exhibits a bfa-like phenotype, restoring flbD-dependent class III/IV transcription in strains that contain mutations in class II flagellar structural genes. Taken together, these results indicated both a positive and negative regulatory function for FliX in coupling the assembly of class II basal body components to gene expression.

Evolution of Microbial Pathogens, 2006

EVOLUTION OF PATHOGENS IN SOIL Rachel Muir and Man-Wah Tan 8 The soil is a particularly complex e... more EVOLUTION OF PATHOGENS IN SOIL Rachel Muir and Man-Wah Tan 8 The soil is a particularly complex environment and is one of the most dynamic sites of biologi-cal interactions in nature. It is not merely a static physicochemical matrix, but a biological system in a ...

Molecular Microbiology, 2002

Mutations in FlbD that relieve the dependency on flagellum assembly alter the temporal and spatia... more Mutations in FlbD that relieve the dependency on flagellum assembly alter the temporal and spatial pattern of developmental transcription in Caulobacter crescentus expression and their capacity to reinitiate chromosomal DNA replication (reviewed in Brun et al., 1994; Gober and Marques, 1995; Wu and Newton, 1997; Gober and England, 2000). For example, the newly formed stalked cell initiates DNA replication almost immediately after cell division, whereas replication is repressed for a period of time in the swarmer cell. Following this period of repression, the flagellum is shed, a stalk is synthesized in its place and DNA replication initiates (Fig. 1). This programme of cellular differentiation is directed, in part, by both cell cycle and spatial transcription which is regulated by members of the large family of bacterial two-component regulatory systems. The basic regulatory paradigm common to these signal transduction systems consists of a stimulatory cue, often environmental, which is sensed by and, in turn, activates autophosphorylation of a sensor histidine kinase (reviewed in Parkinson and Kofoid, 1992). The phosphate from this kinase is then transferred to a conserved receiver domain of a response regulator protein that, very often, is a transcription factor. One hallmark of the C. crescentus programme of cellular differentiation is the temporal, and spatial, biogenesis of a single polar flagellum at the pole opposite the stalk (Fig. 1). The synthesis of this flagellum is regulated by two distinct global-response regulator transcription factors, CtrA and FlbD, that function at specific times and locations in the pre-divisional cell (reviewed in Brun et al., 1994; Gober and Marques, 1995; Wu and Newton, 1997; Gober and England, 2000). The biogenesis of the polar flagellum requires at least 50 gene products and is regulated by a complex transacting hierarchy that is influenced both by progression of the cell cycle and flagellum assembly. The earliest synthesized flagellar components consist of those that encode the MS-ring (fliF), the flagellar switch and components of the flagellum-specific secretory system. These early, class II genes share a conserved promoter sequence that contains a binding site for the transcription factor, CtrA (Quon et al., 1996; Domian et al., 1997; Reisenauer et al., 1999). CtrA is activated by a cell cycle cue, which is presumably linked to the initiation of DNA replication. Following the expression and assembly of the early, class II-encoded flagellar structure, the genes encoding Molecular Microbiology (2002) 43(3), 597-615

Molecular Microbiology, 2001

The first flagellar assembly checkpoint of Caulobacter crescentus couples assembly of the early c... more The first flagellar assembly checkpoint of Caulobacter crescentus couples assembly of the early class II components of the basal body complex to the expression of class III and IV genes, which encode extracytoplasmic structures of the flagellum. The transcription of class III/IV flagellar genes is activated by the response regulator factor, FlbD. Gain of function mutations in flbD, termed bfa, can bypass the transcriptional requirement for the assembly of class II flagellar structures. Here we show that the class II flagellar gene fliX encodes a transacting factor that couples flagellar assembly to FlbD-dependent transcription. We show that the overexpression of fliX can suppress class III/IV gene expression in both wild-type and flbD-bfa cells. Introduction of a bfa allele of flbD into cells possessing a deletion in fliX restores motility indicating that FliX is not a structural component of the flagellum, but rather a transacting factor. Furthermore, extragenic motile suppressors which arise in DfliX cells map to the flbD locus. These results indicate that FlbD functions downstream of FliX in activating class III/IV transcription. b-Lactamase fusions to FliX and analysis of cellular fractions demonstrate that FliX is a cytosolic protein that demonstrates some peripheral association with the cytoplasmic membrane. In addition, we have isolated a mutant allele of fliX that exhibits a bfa-like phenotype, restoring flbD-dependent class III/IV transcription in strains that contain mutations in class II flagellar structural genes. Taken together, these results indicated both a positive and negative regulatory function for FliX in coupling the assembly of class II basal body components to gene expression.

Molecular Microbiology, 2001

Biogenesis of the single polar flagellum of Caulobacter crescentus is regulated by a complex inte... more Biogenesis of the single polar flagellum of Caulobacter crescentus is regulated by a complex interplay of cell cycle events and the progression of flagellum assembly. The expression of class III/IV flagellar genes requires the assembly of an early flagellar basal body structure, encoded by class II genes, and is activated by the transcription factor FlbD. Previous experiments indicated that the class II flagellar gene, flbE, encoded a transacting factor that was required for FlbD activity. Here, using mutant alleles of flbE we have determined that FlbE is either a structural component of the flagellum or is required for flagellar assembly and does not, as originally proposed, function as a transacting factor. We also demonstrate that two deleted derivatives of flbE have a dominant negative effect on the transcriptional activation of class III/IV flagellar genes that can be relieved by a gain-of-function mutation in flbD called bfa. This same mutation in flbD has been shown to restore class III/IV transcription in the absence of early class II flagellar assembly. These deleted mutants of flbE also exhibited a filamentous cell phenotype that was indistinguishable from that previously observed in class II flagellar mutants. Introduction of a flbD-bfa mutation into these cells expressing the deleted alleles of flbE, as well as several class II mutant strains, restored normal cell division and FtsZ localization. These results suggest that class III/IV transcription and a step in cell division are coupled to flagellar assembly by the same genetic pathway.

Molecular Microbiology, 2004

The temporal and spatial transcription of late flagellar genes in Caulobacter crescentus is regul... more The temporal and spatial transcription of late flagellar genes in Caulobacter crescentus is regulated by the s s s s 54 transcriptional activator, FlbD. One requirement for FlbD activity is the assembly of a structure encoded by early, class II flagellar genes. In this report, we show that the transacting factor FliX predominantly functions as a negative regulator of FlbD activity in the absence of the class II-encoded flagellar structure. In contrast, a mutant FliX that bypasses the transcriptional requirement for early flagellar assembly is incapable of repressing FlbD in a class II flagellar mutant. Expression of this mutant allele, fliX1 , does not alter the temporal pattern of FlbD-dependent transcription. Remarkably, this mutation confers the correct cell cycle timing of hook operon transcription in a strain that cannot assemble the flagellum, indicating that the progression of flagellar assembly is a minor influence on temporal gene expression. Using a two-hybrid assay, we present evidence that FliX regulates FlbD through a direct interaction, a novel mechanism for this class of s s s s 54 transcriptional activator. Furthermore, increasing the cellular levels of FliX results in an increase in the concentration of FlbD, and a corresponding increase in FlbD-activated transcription, suggesting that FliX and FlbD form a stable complex in Caulobacter. FliX and FlbD homologues are present in several polar-flagellated bacteria, indicating that these proteins constitute an evolutionarily conserved regulatory pair in organisms where flagellar biogenesis is likely to be under control of the cell division cycle.

Journal of Bacteriology, 2005

In the Caulobacter crescentus predivisional cell, class III and IV flagellar genes, encoding the ... more In the Caulobacter crescentus predivisional cell, class III and IV flagellar genes, encoding the extracytoplasmic components of the flagellum, are transcribed in the nascent swarmer compartment. This asymmetric expression pattern is attributable to the compartmentalized activity of the σ54-dependent transcriptional activator FlbD. Additionally, these temporally transcribed flagellar promoters possess a consensus sequence for the DNA-binding protein integration host factor (IHF), located between the upstream FlbD binding site and the promoter sequences. Here, we deleted the C. crescentus gene encoding the β-subunit of the IHF, ihfB (himD), and examined the effect on flagellar gene expression. The ΔihfB strain exhibited a mild defect in cell morphology and impaired motility. Using flagellar promoter reporter fusions, we observed that expression levels of a subset of class III flagellar promoters were decreased by the loss of IHF. However, one of these promoters, fliK-lacZ, exhibited a...

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, 2007

A yellow-pigmented, Gram-positive, aerobic, non-motile, non-spore-forming, irregular rod-shaped b... more A yellow-pigmented, Gram-positive, aerobic, non-motile, non-spore-forming, irregular rod-shaped bacterium (strain TAN 31504T) was isolated from the bacteriophagous nematode Caenorhabditis elegans. Based on 16S rRNA gene sequence similarity, DNA G+C content of 69.5 mol%, 2,4-diaminobutyric acid in the cell-wall peptidoglycan, major menaquinone MK-11, abundance of anteiso- and iso-fatty acids, polar lipids diphosphatidylglycerol and phosphatidylglycerol and a number of shared biochemical characteristics, strain TAN 31504T was placed in the genus Leucobacter. DNA–DNA hybridization comparisons demonstrated a 91 % DNA–DNA relatedness between strain TAN 31504T and Leucobacter chromiireducens LMG 22506T indicating that these two strains belong to the same species, when the recommended threshold value of 70 % DNA–DNA relatedness for the definition of a bacterial species by the ad hoc committee on reconciliation of approaches to bacterial systematics is considered. Based on distinct differen...

Applied and Environmental Microbiology, 2008

We describe the pathogenic interaction between a newly described gram-positive bacterium, Leucoba... more We describe the pathogenic interaction between a newly described gram-positive bacterium, Leucobacter chromiireducens subsp. solipictus strain TAN 31504, and the nematode Caenorhabditis elegans . TAN 31504 pathogenesis on C. elegans is exerted primarily through infection of the adult nematode uterus. TAN 31504 enters the uterus through the external vulval opening, and the ensuing uterine infection is strongly correlated with a significant reduction in host life span. Young worms can feed and develop on TAN 31504, but not preferably over the standard food source. C. elegans worms reared on TAN 31504 as the sole food source develop into thin adults with little intestinal fat stores, produce few progeny, and subsequently cannot persist on the pathogenic food source. Within 12 h of exposure, adult worms challenged with TAN 31504 alter the expression of a number of C. elegans innate immunity-related genes, including nlp-29 , which encodes a neuropeptide-like protein. C. elegans worms exp...

Evolution of microbial …, 2006

EVOLUTION OF PATHOGENS IN SOIL Rachel Muir and Man-Wah Tan 8 The soil is a particularly complex e... more EVOLUTION OF PATHOGENS IN SOIL Rachel Muir and Man-Wah Tan 8 The soil is a particularly complex environment and is one of the most dynamic sites of biologi-cal interactions in nature. It is not merely a static physicochemical matrix, but a biological system in a ...

International journal of systematic and …, 2007

A yellow-pigmented, Gram-positive, aerobic, non-motile, non-spore-forming, irregular rod-shaped b... more A yellow-pigmented, Gram-positive, aerobic, non-motile, non-spore-forming, irregular rod-shaped bacterium (strain TAN 31504 T) was isolated from the bacteriophagous nematode Caenorhabditis elegans. Based on 16S rRNA gene sequence similarity, DNA G+C content of 69.5 mol%, 2,4-diaminobutyric acid in the cell-wall peptidoglycan, major menaquinone MK-11, abundance of anteiso-and iso-fatty acids, polar lipids diphosphatidylglycerol and phosphatidylglycerol and a number of shared biochemical characteristics, strain TAN 31504 T was placed in the genus Leucobacter. DNA-DNA hybridization comparisons demonstrated a 91 % DNA-DNA relatedness between strain TAN 31504 T and Leucobacter chromiireducens LMG 22506 T indicating that these two strains belong to the same species, when the recommended threshold value of 70 % DNA-DNA relatedness for the definition of a bacterial species by the ad hoc committee on reconciliation of approaches to bacterial systematics is considered. Based on distinct differences in morphology, physiology, chemotaxonomic markers and various biochemical characteristics, it is proposed to split the species L. chromiireducens into two novel subspecies, Leucobacter chromiireducens subsp. chromiireducens subsp. nov. (type strain L-1 T 5CIP 108389 T 5LMG 22506 T) and Leucobacter chromiireducens subsp. solipictus subsp. nov. (type strain TAN 31504 T 5DSM 18340 T 5ATCC BAA-1336 T).

Applied and environmental microbiology, 2008

We describe the pathogenic interaction between a newly described gram-positive bacterium, Leucoba... more We describe the pathogenic interaction between a newly described gram-positive bacterium, Leucobacter chromiireducens subsp. solipictus strain TAN 31504, and the nematode Caenorhabditis elegans. TAN 31504 pathogenesis on C. elegans is ...

Molecular microbiology, 2004

The temporal and spatial transcription of late flagellar genes in Caulobacter crescentus is regul... more The temporal and spatial transcription of late flagellar genes in Caulobacter crescentus is regulated by the s s s s 54 transcriptional activator, FlbD. One requirement for FlbD activity is the assembly of a structure encoded by early, class II flagellar genes. In this report, we show that the transacting factor FliX predominantly functions as a negative regulator of FlbD activity in the absence of the class II-encoded flagellar structure. In contrast, a mutant FliX that bypasses the transcriptional requirement for early flagellar assembly is incapable of repressing FlbD in a class II flagellar mutant. Expression of this mutant allele, fliX1 , does not alter the temporal pattern of FlbD-dependent transcription. Remarkably, this mutation confers the correct cell cycle timing of hook operon transcription in a strain that cannot assemble the flagellum, indicating that the progression of flagellar assembly is a minor influence on temporal gene expression. Using a two-hybrid assay, we present evidence that FliX regulates FlbD through a direct interaction, a novel mechanism for this class of s s s s 54 transcriptional activator. Furthermore, increasing the cellular levels of FliX results in an increase in the concentration of FlbD, and a corresponding increase in FlbD-activated transcription, suggesting that FliX and FlbD form a stable complex in Caulobacter. FliX and FlbD homologues are present in several polar-flagellated bacteria, indicating that these proteins constitute an evolutionarily conserved regulatory pair in organisms where flagellar biogenesis is likely to be under control of the cell division cycle.

Molecular microbiology, 2002

Mutations in FlbD that relieve the dependency on flagellum assembly alter the temporal and spatia... more Mutations in FlbD that relieve the dependency on flagellum assembly alter the temporal and spatial pattern of developmental transcription in Caulobacter crescentus expression and their capacity to reinitiate chromosomal DNA replication (reviewed in Brun et al., 1994; Gober and Marques, 1995; Wu and Newton, 1997; Gober and England, 2000). For example, the newly formed stalked cell initiates DNA replication almost immediately after cell division, whereas replication is repressed for a period of time in the swarmer cell. Following this period of repression, the flagellum is shed, a stalk is synthesized in its place and DNA replication initiates (Fig. 1). This programme of cellular differentiation is directed, in part, by both cell cycle and spatial transcription which is regulated by members of the large family of bacterial two-component regulatory systems. The basic regulatory paradigm common to these signal transduction systems consists of a stimulatory cue, often environmental, which is sensed by and, in turn, activates autophosphorylation of a sensor histidine kinase (reviewed in Parkinson and Kofoid, 1992). The phosphate from this kinase is then transferred to a conserved receiver domain of a response regulator protein that, very often, is a transcription factor. One hallmark of the C. crescentus programme of cellular differentiation is the temporal, and spatial, biogenesis of a single polar flagellum at the pole opposite the stalk (Fig. 1). The synthesis of this flagellum is regulated by two distinct global-response regulator transcription factors, CtrA and FlbD, that function at specific times and locations in the pre-divisional cell (reviewed in Brun et al., 1994; Gober and Marques, 1995; Wu and Newton, 1997; Gober and England, 2000). The biogenesis of the polar flagellum requires at least 50 gene products and is regulated by a complex transacting hierarchy that is influenced both by progression of the cell cycle and flagellum assembly. The earliest synthesized flagellar components consist of those that encode the MS-ring (fliF), the flagellar switch and components of the flagellum-specific secretory system. These early, class II genes share a conserved promoter sequence that contains a binding site for the transcription factor, CtrA (Quon et al., 1996; Domian et al., 1997; Reisenauer et al., 1999). CtrA is activated by a cell cycle cue, which is presumably linked to the initiation of DNA replication. Following the expression and assembly of the early, class II-encoded flagellar structure, the genes encoding Molecular Microbiology (2002) 43(3), 597-615

Molecular microbiology, 2001

The first flagellar assembly checkpoint of Caulobacter crescentus couples assembly of the early c... more The first flagellar assembly checkpoint of Caulobacter crescentus couples assembly of the early class II components of the basal body complex to the expression of class III and IV genes, which encode extracytoplasmic structures of the flagellum. The transcription of class III/IV flagellar genes is activated by the response regulator factor, FlbD. Gain of function mutations in flbD, termed bfa, can bypass the transcriptional requirement for the assembly of class II flagellar structures. Here we show that the class II flagellar gene fliX encodes a transacting factor that couples flagellar assembly to FlbD-dependent transcription. We show that the overexpression of fliX can suppress class III/IV gene expression in both wild-type and flbD-bfa cells. Introduction of a bfa allele of flbD into cells possessing a deletion in fliX restores motility indicating that FliX is not a structural component of the flagellum, but rather a transacting factor. Furthermore, extragenic motile suppressors which arise in DfliX cells map to the flbD locus. These results indicate that FlbD functions downstream of FliX in activating class III/IV transcription. b-Lactamase fusions to FliX and analysis of cellular fractions demonstrate that FliX is a cytosolic protein that demonstrates some peripheral association with the cytoplasmic membrane. In addition, we have isolated a mutant allele of fliX that exhibits a bfa-like phenotype, restoring flbD-dependent class III/IV transcription in strains that contain mutations in class II flagellar structural genes. Taken together, these results indicated both a positive and negative regulatory function for FliX in coupling the assembly of class II basal body components to gene expression.