Characterization of rcsB and rcsC from Escherichia coli O9:K30:H12 and examination of the role of the rcs regulatory system in expression of group I capsular polysaccharides

Jayaratne, Padman; Keenleyside, Wendy; MacLachlan, P R; Dodgson, C; Whitfield, Chris

doi:10.1128/jb.175.17.5384-5394.1993

Cited by 62 publications

(65 citation statements)

References 58 publications

(52 reference statements)

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“…The histidine kinase-like domain of CyaC (Leu-511 to Leu-729) is similar to the transmitter domain of histidine kinase proteins. This region of CyaC shows 27.9% identity to the LemA protein of Pseudomonas syringae (23), 32.4% identity to the PhoR protein of Shigella dysenteriae (30), 26.0% identity to the RcsC protein of E. coli (24), and 33.3% identity to the PleC protein of Caulobacter crescentus (50). The highly conserved histidine residue of histidine kinase proteins and the aspartate residue of response regulator proteins, both of which are responsible for protein phosphorylation, exist in regions of CyaC (His-524, Asp-59, and Asp-847).…”

Section: Resultsmentioning

confidence: 99%

Isolation and characterization of multiple adenylate cyclase genes from the cyanobacterium Anabaena sp. strain PCC 7120

Katayama

Ohmori

1997

J Bacteriol

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Adenylate cyclase genes, designated cyaA, cyaB1, cyaB2, cyaC, and cyaD, were isolated from the filamentous cyanobacterium Anabaena sp. strain PCC 7120 by complementation of a strain of Escherichia coli defective for the presence of cya. These genes encoded polypeptides consisting of 735, 859, 860, 1,155, and 546 amino acid residues, respectively. Deduced amino acid sequences of the regions near the C-terminal ends of these cya genes were similar to those of catalytic domains of eukaryotic adenylate cyclases. The remaining part of each cya gene towards its N-terminal end showed a characteristic structure. CyaA had two putative membrane-spanning regions. Both CyaB1 and CyaB2 had regions that were very similar to the cyclic GMP (cGMP)-binding domain of cGMP-stimulated cGMP phosphodiesterase. CyaC consisted of four distinct domains forming sequentially from the N terminus: a response regulator-like domain, a histidine kinase-like domain, a response regulatorlike domain, and the catalytic domain of adenylate cyclase. CyaD contained the forkhead-associated domain in its N-terminal region. Expression of these genes was examined by reverse transcription-PCR. The transcript of cyaC was shown to be predominant in this cyanobacterium. The cellular cyclic AMP level in the disruptant of the cyaC mutant was much lower than that in the wild type.Cyclic AMP (cAMP) regulates various biological activities in both prokaryotes and eukaryotes. In prokaryotes, cAMP and cAMP receptor protein regulate gene expression (7). In eukaryotes, cAMP regulates enzyme activity (11), channel activity (42), and gene expression (34) via the cAMP-dependent protein kinase. In cyanobacteria, it has been reported that cellular cAMP content is affected by growth conditions (17,22), that cAMP concentration changes rapidly in response to light-off and light-on signals (37) or to a pH shift (36), and that cAMP interferes with the pattern formation of heterocysts (43). However, it is still unknown whether cAMP functions as an intracellular signal molecule in cyanobacteria.Recently, we isolated adenylate cyclase (cya) genes from the filamentous cyanobacteria Anabaena cylindrica (26) and Spirulina platensis (52). Regions near the C-terminal ends of the adenylate cyclases of both cyanobacteria showed significant structural similarity to the catalytic domain of the adenylate cyclase of eukaryotes but showed no homology to that of prokaryotes such as Escherichia coli. Furthermore, cyanobacterial adenylate cyclases appeared to be membrane oriented.To study the physiological functions of these adenylate cyclases, disruption or overexpression of cya genes is indispensable. Unfortunately, both A. cylindrica and S. platensis are not transformable, so they are not suitable for this purpose. In this study, we isolated cya genes from a transformable cyanobacterium, Anabaena sp. strain PCC 7120, and constructed mutants defective in cya genes. Some physiological properties of these mutants were determined. MATERIALS AND METHODSStrains and growth conditions. Anabaena sp. s...

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Section: Resultsmentioning

confidence: 99%

Isolation and characterization of multiple adenylate cyclase genes from the cyanobacterium Anabaena sp. strain PCC 7120

Katayama

Ohmori

1997

J Bacteriol

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“…Furthermore, neither an rcsB null mutation nor rcsB overexpression affected expression of the group I capsuleencoding (cps) operon. 114 This suggested that the effect of rcsB overexpression on group I capsule production was indirect. Indeed, it was found that rcsB overexpression induced the galF gene involved in producing sugar nucleotide precursors, and that there was a putative RcsAB-binding sequence (RcsAB box) upstream of galF.…”

Section: 21109mentioning

confidence: 99%

“…In some E. coli strains the production of group I capsule was induced by overexpression of rcsB. 114 However, an rcsB null mutation did not affect capsule production. Furthermore, neither an rcsB null mutation nor rcsB overexpression affected expression of the group I capsuleencoding (cps) operon.…”

Section: 21109mentioning

confidence: 99%

Regulation of bacterial virulence gene expression by cell envelope stress responses

Flores-Kim

Darwin

2014

Virulence

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“…The inactivation of the lon gene resulted in overproduction of amylovoran. In E. coli and E. stewartii, the gene coding for the second positive regulator RcsB is localized adjacent to the rcsC gene (16,31,46). The two genes are transcribed in opposite directions, and the coded proteins are proposed to be part of a two-component regulatory system.…”

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Characterization of the rcsB gene from Erwinia amylovora and its influence on exoploysaccharide synthesis and virulence of the fire blight pathogen

Bereswill

Geider

1997

J Bacteriol

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RcsB belongs to a family of positive regulators of exopolysaccharide synthesis in various enterobacteria. The rcsB gene of the fire blight pathogen Erwinia amylovora was cloned by PCR amplification with consensus primers, and its role in exopolysaccharide (EPS) synthesis was investigated. Its overexpression from highcopy-number plasmids stimulated the synthesis of the acidic EPS amylovoran and suppressed expression of the levan-forming enzyme levansucrase. Inactivation of rcsB by site-directed mutagenesis created mutants that were deficient in amylovoran synthesis and avirulent on host plants. In addition, a cosmid which complemented rcsB mutants was selected from a genomic library. The spontaneous E. amylovora mutant E8 has a similar phenotype and was complemented by the cloned rcsB gene. The rcsB region of strain E8 was also amplified by PCR, and the mutation was characterized as a nine-nucleotide deletion at the start of the rcsB gene. Nucleotide sequence analysis of the E. amylovora rcsB region and the predicted amino acid sequence of RcsB revealed extensive homology to rcsB and the encoded protein of other bacteria such as Escherichia coli and Erwinia stewartii. In all three organisms, rcsB is localized adjacent to the rcsC gene, which is transcribed in the opposite direction of rcsB. The E. amylovora rcsB gene has now been shown to strongly affect the formation of disease symptoms of a plant pathogen.The gram-negative bacterium Erwinia amylovora causes fire blight on rosaceous plants. Typical symptoms are wilting, necrotic lesions, and production of ooze which consists mainly of capsular exopolysaccharide (EPS) produced by the bacteria. Production of capsule polysaccharides is a common feature of many gram-negative bacteria (49), especially plant pathogens (32). The capsule creates a favorable microenvironment for the bacterial cells and protects them against water stress and plant defense mechanisms. E. amylovora produces two types of EPS, amylovoran and levan (2). Levan is a neutral homopolymer composed of ␤-2,6-linked fructose molecules, and its synthesis is mediated by the enzyme levansucrase. Levansucrase is secreted into the environment and uses sucrose as a substrate (29). The levansucrase gene (lsc) of E. amylovora was recently cloned and characterized and was found to be constitutively expressed by the pathogen (24). Amylovoran is an acidic heteropolymer composed of a pentasaccharide repeating unit that contains four galactose molecules and one glucuronic acid residue (35,45). Its synthesis is encoded by the ams (for amylovoran synthesis) operon, which was also cloned and sequenced (13). The 12 genes amsA to amsL are organized in a large gene cluster on the bacterial chromosome. Transposonand site-directed mutagenesis of the lsc gene and the ams operon revealed that both types of EPS are important for the virulence of E. amylovora. Mutants that lack the synthesis of levan were reduced in virulence (24), and mutants deficient in amylovoran synthesis were nonpathogenic (4, 9). The precise role of EP...

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Characterization of rcsB and rcsC from Escherichia coli O9:K30:H12 and examination of the role of the rcs regulatory system in expression of group I capsular polysaccharides

Abstract: In Escherichia coil K-12, RcsC and RcsB are thought to act as the sensor and effector components, respectively, of a two-component regulatory system which regulates expression of the slime polysaccharide colanic acid (V. Stout and S. Gottesman, J. Bacteriol. 172:659-669, 1990

Cited by 62 publications

References 58 publications

Isolation and characterization of multiple adenylate cyclase genes from the cyanobacterium Anabaena sp. strain PCC 7120

Isolation and characterization of multiple adenylate cyclase genes from the cyanobacterium Anabaena sp. strain PCC 7120

Regulation of bacterial virulence gene expression by cell envelope stress responses

Characterization of the rcsB gene from Erwinia amylovora and its influence on exoploysaccharide synthesis and virulence of the fire blight pathogen

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