Nucleotide sequence of the Rickettsia prowazekii citrate synthase gene

Abstract

The Rickettsia prowazekii citrate synthase (gitA) gene, previously cloned in Escherichia coli, was localized to a 2.0-kilobase chromosomnal fragment. DNA sequence analysis of a portion of this fragment revealed an open reading frame of 1,308 base pafrs that encodes a protein of 435 amino acids with a molecular weight of 49,171. This translation product is comparable in size to both the E. coli Snd pig heart citrate synthase monomers and to the protein synthesized in E. coli minicells containing the rickettsial gene. Comparisons between the deduced amino acid sequence of R. prowazekii citrate synthase and those of the E. coli and pig heart enzymes revealed extensive homology (59%) between the two bacterial proteins. In contrast, only 20% of the rickettsial enzyme residues were shared with the functionally similar pig heart enzyine residUes. Upstream from the open reading frame and in close proximity to one anothet, sequences with homology to E. coli consensus sequences for RNA polymerase and ribosome binding were identified. S1 nuclease mapping experiments demobstrated that the start of transcription for this gene in E. coli was located in the upstream region. Codon usage in the rickettsial git4 gene was found to be very biased and differed from the pattern observed in E. coli. Adenine and uracil were used preferentially in the third base position of rickettsial codons.Rickettsia prowazekii is an obligate intracellular parasitic bacterium that multiplies within the cytoplasm of its eucaryotic host cell rather than within a phagosome or phagolysosome. Its exploitation of this environment is reflected in a variety of novel features, most notably the existence of an ADP-ATP transport system that permits the rickettsiae to accumulate ATP directly from the cytoplasm of the host cell (44). However, the rickettsiae are not strict energy parasites and can generate their own ATP via the tricarboxylic acid cycle and oxidative phosphorylation (39). This ability is probably necessary for cell viability during periods when the host cell ATP reserves are depleted or during transit of the bacterium to a new host cell. Central to the regulation of the tricarboxylic acid cycle is the enzyme citrate synthase.Citrate synthase, the first enzyme of the tricarboxylic acid cycle, has been extensively studied in a variety of procaryotic and. eucaryotic cells (30,, 41, 43). These studies have identified two main types of citrate synthase: a "small" type (molecular weight, -106,000) found in eucaryotic cells and gram-positive bacteria and a "large" type (molecular weight, -250,000) found exclusively in gram-negative bacteria. The small enzyme is a dimer composed of two identical subunits, while the large enzyme is a multimer composed of four to six identical subunits. The two types of enzyme also differ in their sensitivity to certain effectors. The small enzyme is senlsitive in vitro to inhibition by ATP but not by at-ketoglutarate or NADH, while the large enzyme is sensitive in vitro to inhibition by a-ketoglutarate and NADH but not by ...

Citrate synthase from the thermophilic archaebacteria Thermoplasma acidophilum and Sulfolobus acidocaldarius

“…Moreover, the region which shows no homology (residues l-9 of the Thermoplasma enzyme) is also non-homologous when the eukaryotic and eubacterial enzymes are aligned [22]. Secondly, the alignment in fig.1 indicates that the archaebacterial enzyme will be approx.…”

“…E. coli [19,20], Acinetobacter anitratum [21] and Rickettsia prowazekii [22]. For We have been able to compare the limited sequence with that of pig heart citrate synthase on the basis that the threedimensional structure of the eukaryotic enzyme is known [17] and therefore the search for sequence homology can incorporate penalties for insertions and deletions in regions of secondary structure.…”

“…Identical residues between the two sequences are boxed with solid lines and conservative changes with dotted lines. The corresponding sequences of the citrate synthases from yeast, E. coli, A. anitratum and R. prowazekii are aligned according to [21,22], based on whole sequence homologies.…”

Detección de rickettsia rickettsii brump (RICKETTSIALES: RICKETTSIACEAE) en la garrapata café del perro rhipicephalus sanguineus latreille (IXODIDA: IXODIDAE) en la comarca lagunera, zona reemergente de fiebre manchada en México

“…La calidad y cantidad de ADN obtenido por medio de nanodrop (Thermo Scientific® NanoDrop 2000) fue evaluada, resuspendiéndola en 20 µl de buffer TE 1X [400 µl EDTA (Life Invitrogen®, USA) + 4 ml Tris Base (Bio Basic Canada Inc ®] para almacenarla a -20 ºC. Para las pruebas de PCR se utilizó el iniciador genérico que amplifica el gen gltA en un rango de 380-397 pb (Wood et al, 1987) para Ricketssia spp: Forward: RpCS.877p GGGGGCCTGCTCACGGCGG, Reverse: RpCS.1258n ATTGCAAAAAGTACAGT-GAACA (Regnery et al, 1991). La reacción de PCR se realizó con la siguiente mezcla de reacción: MgCl 2 a una concentración de 1.5 mM, buffer 10X (200 mM Tris-HCl pH 8.4 Bio Basic Canada Inc ®), dNTP´s (Life Invitrogen ®, USA) a una concentración de 0.2 mM, 1.25 U de Taq DNA polimerasa (Life Invitrogen ®, USA), ADN templado concentrado a 20-25 ng/µl, 20 pmol/ µl de cada uno de los iniciadores (RpCS.877p, RpCS.1258n) y agua milli-Q (Life Invitrogen ®, USA) para completar un Rx de 25 µl.…”

Detection of Brazilian spotted fever infection by polymerase chain reaction in a patient from the state of São Paulo

“…Infection of Vero cells was monitored by immunofluorescence reaction prepared with R. rickettsii-positive human serum, which permitted us to observe the presence of fluorescent microorganisms in form of intracellular bacteria (Marrero & Raoult 1989 Prior to DNA extraction through the use of phenol/ phenol-chloroform, the frozen blood clot was incubated at 56ºC for 2 h for rickettsiae inactivation (Tzianabos et al 1989). Rickettsial DNA was detected by polymerase chain reaction (PCR) using previously described conditions (Regnery et al 1991) and the three sets of primers (GIBCO BRL), RpCs.877p (5'-GGGGGCCTGCTCACGGCGG) and RpCs.1258n (5'-ATTGCAAAAAGTACAGTGAACA) to amplify a 381-bp fragment of the citrate synthase gene (gltA) of Rickettsia species (Wood et al 1987); Rr190.70p (5'-ATGGCGAATATTTCTCCAAAA) and Rr190.602n (5'-AGTGCAGCATTCGCTCCCCCT) for a 532-bp fragment of the 190-kDa surface protein gene (ompA) of SFG rickettsia (Regnery et al 1991); and BG1-21 (5'-GGCAATTAATATCGCTGACGG) and BG2-20 (5'-GCA TCTGCACTAGCACTTTC) for a 650-bp fragment of the 120 kDa surface protein gene (ompB) of SFG and Typho Group rickettsiae (Eremeeva et al 1994). PCR products to be sequenced were cloned into plasmid vector pGEMTEasy (Promega).…”