scholarly journals Molecular characterization and analysis of a gene encoding the acidic repeat protein (Arp) of Treponema pallidum

2007 ◽  
Vol 56 (6) ◽  
pp. 715-721 ◽  
Author(s):  
Hsi Liu ◽  
Berta Rodes ◽  
Robert George ◽  
Bret Steiner
Keyword(s):  
Synthetic Peptides ◽  
Treponema Pallidum ◽  
Signal Peptidase ◽  
Type I ◽  
Type Ii ◽  
Gene Encoding ◽  
Western Blot Assay ◽  
Repeat Protein ◽  
Signal Peptidase I ◽  
Cdc 2

The acidic repeat protein (arp) genes from three subspecies of the treponeme Treponema pallidum (T. pallidum subsp. pallidum, Nichols strain; T. pallidum subsp. pertenue, CDC-1 and CDC-2 strains; and T. pallidum subsp. endemicum, Bosnia A strain) were cloned and sequenced. The predicted protein sequence contained a high percentage of glutamic acid, hence the name acidic repeat protein, or Arp. The protein had a potential membrane-spanning domain and a signal peptidase I site. The gene from the Nichols strain of T. pallidum subsp. pallidum contained a set of 14 nearly identical repeats of a 60 bp sequence, which occupied ∼51 % of the length of the gene. Analyses of arp from laboratory strains showed that the 5′ and 3′ ends of the genes were conserved, but there was considerable heterogeneity in the number of repeats of this 60 bp sequence. Based on amino acid variations, the 14 sequence repeats could be classified into three types, which were named type I, type II and type III repeats. The type II repeat was the most common in the strains examined. The arp gene of the Nichols strain was subsequently cloned into the expression vector pBAD/TOPO ThioFusion. The expressed protein was detected in a Western blot assay using rabbit immune sera produced against T. pallidum, or synthetic peptides derived from the repeat sequences. Using an ELISA, rapid plasma reagin (RPR) test-positive sera reacted with synthetic peptides derived from the repeat region but not with peptides derived from N and C termini of the Arp protein. These results show that the Arp protein is immunogenic and could prove to be a useful target for serological diagnosis of T. pallidum infection.

scholarly journals Gut SymbiontBacteroides fragilisSecretes a Eukaryotic-Like Ubiquitin Protein That Mediates Intraspecies Antagonism

mBio ◽  
2017 ◽  
Vol 8 (6) ◽  
Author(s):  
Maria Chatzidaki-Livanis ◽  
Michael J. Coyne ◽  
Kevin G. Roelofs ◽  
Rahul R. Gentyala ◽  
Jarreth M. Caldwell ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Bacteroides Fragilis ◽  
Signal Peptidase ◽  
Metagenomic Data ◽  
Antimicrobial Protein ◽  
Secretion Systems ◽  
Human Gut ◽  
Gene Encoding ◽  
Toxic Activity ◽  
Signal Peptidase I

ABSTRACTHuman gutBacteroidesspecies produce different types of toxins that antagonize closely related members of the gut microbiota. Some are toxic effectors delivered by type VI secretion systems, and others are non-contact-dependent secreted antimicrobial proteins. Many strains ofBacteroides fragilissecrete antimicrobial molecules, but only one of these toxins has been described to date (Bacteroidalessecreted antimicrobial protein 1 [BSAP-1]). In this study, we describe a novel secreted protein produced byB. fragilisstrain 638R that mediated intraspecies antagonism. Using transposon mutagenesis and deletion mutation, we identified a gene encoding a eukaryotic-like ubiquitin protein (BfUbb) necessary for toxin activity against a subset ofB. fragilisstrains. The addition ofubbinto a heterologous background strain conferred toxic activity on that strain. We found this gene to be one of the most highly expressed in theB. fragilisgenome. The mature protein is 84% similar to human ubiquitin but has an N-terminal signal peptidase I (SpI) signal sequence and is secreted extracellularly. We found that the mature 76-amino-acid synthetic protein has very potent activity, confirming that BfUbb mediates the activity. Analyses of human gut metagenomic data sets revealed thatubbis present in 12% of the metagenomes that have evidence ofB. fragilis. As 638R produces both BSAP-1 and BfUbb, we performed a comprehensive analysis of the toxin activity of BSAP-1 and BfUbb against a set of 40B. fragilisstrains, revealing that 75% ofB. fragilisstrains are targeted by one or the other of these two secreted proteins of strain 638R.IMPORTANCEWe are just beginning to understand some of the important interactions that occur between microbes of the human gut microbiota that dictate the composition and abundance of its constituent members. The ability of one member to produce molecules that directly kill a coresident member has been shown among minor gut species and is just starting to be studied in the abundantBacteroidesspecies. Here, we show that some strains ofBacteroides fragilishave acquired a gene encoding a secreted eukaryotic-like ubiquitin protein with potent inhibitory activity against otherB. fragilisstains. This is the first bacterially encoded ubiquitin-like molecule shown to function like a bacterial toxin. This molecule is an example of a gut symbiont acquiring and adapting a eukaryotic molecule likely to increase its competitiveness in the mammalian gut. Understanding antagonistic factors produced by abundant gut symbionts is an important prerequisite to properly engineer strains to colonize the gut for health benefits.

Analysis of a Streptococcus pneumoniae gene encoding signal peptidase I and overproduction of the enzyme

Gene ◽  
1997 ◽  
Vol 194 (2) ◽  
pp. 249-255 ◽  
Author(s):  
Yian-Biao Zhang ◽  
Bill Greenberg ◽  
Sanford A Lacks
Keyword(s):  
Streptococcus Pneumoniae ◽  
Signal Peptidase ◽  
Gene Encoding ◽  
Signal Peptidase I

scholarly journals The lspA Gene, Encoding the Type II Signal Peptidase of Rickettsia typhi: Transcriptional and Functional Analysis

2006 ◽  
Vol 189 (2) ◽  
pp. 336-341 ◽  
Author(s):  
M. Sayeedur Rahman ◽  
Shane M. Ceraul ◽  
Sheila M. Dreher-Lesnick ◽  
Magda S. Beier ◽  
Abdu F. Azad
Keyword(s):  
Amino Acid Sequences ◽  
Signal Peptidase ◽  
Secretory Proteins ◽  
Transcriptional Level ◽  
Temperature Sensitive ◽  
Type I ◽  
Type Ii ◽  
Intracellular Growth ◽  
Rickettsia Typhi ◽  
Differential Expression Pattern

ABSTRACT Lipoprotein processing by the type II signal peptidase (SPase II) is known to be critical for intracellular growth and virulence for many bacteria, but its role in rickettsiae is unknown. Here, we describe the analysis of lspA, encoding a putative SPase II, an essential component of lipoprotein processing in gram-negative bacteria, from Rickettsia typhi. Alignment of deduced amino acid sequences shows the presence of highly conserved residues and domains that are essential for SPase II activity in lipoprotein processing. The transcription of lspA, lgt (encoding prolipoprotein transferase), and lepB (encoding type I signal peptidase), monitored by real-time quantitative reverse transcription-PCR, reveals a differential expression pattern during various stages of rickettsial intracellular growth. The higher transcriptional level of all three genes at the preinfection time point indicates that only live and metabolically active rickettsiae are capable of infection and inducing host cell phagocytosis. lspA and lgt, which are involved in lipoprotein processing, show similar levels of expression. However, lepB, which is involved in nonlipoprotein secretion, shows a higher level of expression, suggesting that LepB is the major signal peptidase for protein secretion and supporting our in silico prediction that out of 89 secretory proteins, only 14 are lipoproteins. Overexpression of R. typhi lspA in Escherichia coli confers increased globomycin resistance, indicating its function as SPase II. In genetic complementation, recombinant lspA from R. typhi significantly restores the growth of temperature-sensitive E. coli Y815 at the nonpermissive temperature, supporting its biological activity as SPase II in prolipoprotein processing.

Sequence and expression of a type II keratin, K5, in human epidermal cells

1988 ◽  
Vol 8 (1) ◽  
pp. 486-493
Author(s):  
R Lersch ◽  
E Fuchs
Keyword(s):  
Human Gene ◽  
Basal Layer ◽  
Epidermal Cells ◽  
Amino Acid Sequences ◽  
Type I ◽  
Type Ii ◽  
Gene Encoding ◽  
Human Epidermal Cells ◽  
Rna Blot Analysis

We report here the cDNA and amino acid sequences of a human 58-kilodalton type II keratin, K5, which is coexpressed with a 50-kilodalton type I keratin partner, K14, in stratified squamous epithelia. Using a probe specific for the 3'-noncoding portion of this K5 cDNA, we demonstrated the existence of a single human gene encoding this sequence. Using Northern (RNA) blot analysis and in situ hybridization with cRNA probes for both K5 and K14, we examined the expression of these mRNAs in the epidermis and in cultured epidermal cells. Our results indicate that the mRNAs for K5 and K14 are coordinately expressed and abundant in the basal layer of the epidermis. As cells undergo a commitment to terminally differentiate, the expression of both mRNAs seems to be downregulated.

scholarly journals Molecular and Functional Analysis of the lepB Gene, Encoding a Type I Signal Peptidase from Rickettsia rickettsii and Rickettsia typhi

2003 ◽  
Vol 185 (15) ◽  
pp. 4578-4584 ◽  
Author(s):  
M. Sayeedur Rahman ◽  
Jason A. Simser ◽  
Kevin R. Macaluso ◽  
Abdu F. Azad
Keyword(s):  
Spotted Fever ◽  
Rocky Mountain ◽  
Amino Acid Sequences ◽  
Signal Peptidase ◽  
Type I ◽  
Rocky Mountain Spotted Fever ◽  
Rickettsia Typhi ◽  
Rickettsia Rickettsii ◽  
Signal Peptidase I

ABSTRACT The type I signal peptidase lepB genes from Rickettsia rickettsii and Rickettsia typhi, the etiologic agents of Rocky Mountain spotted fever and murine typhus, respectively, were cloned and characterized. Sequence analysis of the cloned lepB genes from R. rickettsii and R. typhi shows open reading frames of 801 and 795 nucleotides, respectively. Alignment analysis of the deduced amino acid sequences reveals the presence of highly conserved motifs that are important for the catalytic activity of bacterial type I signal peptidase. Reverse transcription-PCR and Northern blot analysis demonstrated that the lepB gene of R. rickettsii is cotranscribed in a polycistronic message with the putative nuoF (encoding NADH dehydrogenase I chain F), secF (encoding protein export membrane protein), and rnc (encoding RNase III) genes in a secF-nuoF-lepB-rnc cluster. The cloned lepB genes from R. rickettsii and R. typhi have been demonstrated to possess signal peptidase I activity in Escherichia coli preprotein processing in vivo by complementation assay.

Sensitive immunoassays for the autoantibodies reacting against citrullinated carboxy-terminal telopeptides of type I and type II collagens in patients with rheumatoid arthritis

2005 ◽  
Vol 43 (12) ◽  
Author(s):  
Marja-Kaisa Koivula ◽  
Jarmo Ramberg ◽  
Sari Åman ◽  
Anna Karjalainen ◽  
Markku Hakala ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Synthetic Peptides ◽  
Type I ◽  
Type Ii ◽  
Sensitive Assay ◽  
Chemiluminescence Method ◽  
Assay Methods ◽  
Carboxy Terminal

AbstractWe developed sensitive assay methods for autoantibodies recognizing the citrullinated synthetic peptides derived from type I and type II collagens in patients with rheumatoid arthritis (RA). These peptides were tested with the chemiluminescence method (Nichols Advantage

scholarly journals Sequence and expression of a type II keratin, K5, in human epidermal cells.

1988 ◽  
Vol 8 (1) ◽  
pp. 486-493 ◽  
Author(s):  
R Lersch ◽  
E Fuchs
Keyword(s):  
Human Gene ◽  
Basal Layer ◽  
Epidermal Cells ◽  
Amino Acid Sequences ◽  
Type I ◽  
Type Ii ◽  
Gene Encoding ◽  
Human Epidermal Cells ◽  
Rna Blot Analysis

We report here the cDNA and amino acid sequences of a human 58-kilodalton type II keratin, K5, which is coexpressed with a 50-kilodalton type I keratin partner, K14, in stratified squamous epithelia. Using a probe specific for the 3'-noncoding portion of this K5 cDNA, we demonstrated the existence of a single human gene encoding this sequence. Using Northern (RNA) blot analysis and in situ hybridization with cRNA probes for both K5 and K14, we examined the expression of these mRNAs in the epidermis and in cultured epidermal cells. Our results indicate that the mRNAs for K5 and K14 are coordinately expressed and abundant in the basal layer of the epidermis. As cells undergo a commitment to terminally differentiate, the expression of both mRNAs seems to be downregulated.

scholarly journals Mechanism of Action of the Arylomycin Antibiotics and Effects of Signal Peptidase I Inhibition

2012 ◽  
Vol 56 (10) ◽  
pp. 5054-5060 ◽  
Author(s):  
Peter A. Smith ◽  
Floyd E. Romesberg
Keyword(s):  
Secretory Pathway ◽  
Signal Peptidase ◽  
Model Organisms ◽  
Type I ◽  
Content Type ◽  
E Coli ◽  
Secretion Pathway ◽  
Secretion Stress ◽  
Signal Peptidase I ◽  
Essential Enzyme

ABSTRACTClinically approved antibiotics inhibit only a small number of conserved pathways that are essential for bacterial viability, and the physiological effects of inhibiting these pathways have been studied in great detail. Likewise, characterizing the effects of candidate antibiotics that function via novel mechanisms of action is critical for their development, which is of increasing importance due to the ever-growing problem of resistance. The arylomycins are a novel class of natural-product antibiotics that act via the inhibition of type I signal peptidase (SPase), which is an essential enzyme that functions as part of the general secretory pathway and is not the target of any clinically deployed antibiotic. Correspondingly, little is known about the effects of SPase inhibition or how bacteria may respond to mitigate the associated secretion stress. Using genetically sensitizedEscherichia coliandStaphylococcus aureusas model organisms, we examine the activity of arylomycin as a function of its concentration, bacterial cell density, target expression levels, and bacterial growth phase. The results reveal that the activity of the arylomycins results from an insufficient flux of proteins through the secretion pathway and the resulting mislocalization of proteins. Interestingly, this has profoundly different effects onE. coliandS. aureus. Finally, we examine the activity of arylomycin in combination with distinct classes of antibiotics and demonstrate that SPase inhibition results in synergistic sensitivity when combined with an aminoglycoside.

scholarly journals Diversity of active root-associated methanotrophs of three emergent plants in a eutrophic wetland in northern China 

2020 ◽  
Author(s):  
Jing Cui ◽  
Ji Zhao ◽  
Zheng Wang ◽  
Weiwei Cao ◽  
Shaohua Zhang ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Vegetation Type ◽  
Northern China ◽  
Type I ◽  
Type Ii ◽  
Dominant Group ◽  
Gene Encoding ◽  
Reverse Transcription Pcr ◽  
Scirpus Triqueter ◽  
Aerobic Methanotrophs

Abstract Root-associated aerobic methanotrophs play an important role in regulating methane emissions from the wetlands. However, the influences of the plant genotype on root-associated methanotrophic structures, especially on active flora, remain poorly understood. Transcription of the pmoA gene, encoding particulate methane monooxygenase in methanotrophs, was analyzed by reverse transcription PCR (RT-PCR) of mRNA isolated from root samples of three emergent macrophytes, including Phragmites australis, Typha angustifolia, and Schoenoplectus triqueter (syn. Scirpus triqueter L.) from a eutrophic wetland. High-throughput sequencing of pmoA based on DNA and cDNA was used to analyze the methanotrophic community. Sequencing of cDNA pmoA amplicons confirmed that the structure of active methanotrophic was not always consistent with DNA. A type I methanotroph, Methylomonas, was the most active group in P. australis, whereas Methylocystis, a type II methanotroph, was the dominant group in S. triqueter. In T. angustifolia, these two types of methanotroph existed in similar proportions. However, at the DNA level, Methylomonas was predominant in the roots of all three plants. In addition, vegetation type could have a profound impact on root-associated methanotrophic community at both DNA and cDNA levels. These results indicate that members of the genera Methylomonas (type I) and Methylocystis (type II) can significantly contribute to aerobic methane oxidation in a eutrophic wetland.Key points1. Root-associated Methylomonas was predominant in three macrophytes using DNA approach. 2. Active Methylocystis was dominant in genera Typha and Schoenoplectus, but not in Phragmites. 3. Plant species impact on methanotrophic communities in both DNA and cDNA levels.

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