586 research outputs found
How proteins are transported into mitochondria
Most mitochondrial polypeptides are synthesized outside the organelle as precursors which are usually larger than the ‘mature’ polypeptides found within mitochondria. The precursors are imported into the mitochondria by a process which is independent of protein synthesis but dependent on high-energy phosphate bonds inside the mitochondria. This mechanism is basically different from that which governs the movement of secretory polypeptides across the membrane of the endoplasmic reticulum
Regulation and symbiotic significance of nodulation outer proteins secretion in Sinorhizobium fredii HH103
In this work we show that the Sinorhizobium fredii HH103 ttsI gene is essential for the expression
of the tts genes and secretion of nodulation outer proteins (Nops). Moreover, we demonstrate
for the first time, to our knowledge, that the nod box preceding ttsI is necessary for Nops
secretion. TtsI is responsible for the transcriptional activation of nopX, nopA, rhcJ and rhcQ. We
confirm that the S. fredii HH103 ttsI gene is activated by NodD1 and repressed by NolR. In
contrast, NodD2 is not involved in the regulation of ttsI expression. Despite the dependence of
expression of both ttsI and nodA on NodD1 and flavonoids, clear differences in the capacity of
some flavonoids to activate these genes were found. The expression of the ttsI and nodA
genes was also sensitive to differences in the pH of the media. Secretion of Nops in the ttsI
mutant could not be complemented with a DNA fragment containing the ttsI gene and its nod
box, but it was restored when a plasmid harbouring the ttsI, rhcC2 and y4xK genes was
transferred to the mutant strain. The symbiotic effect of Nops secretion was host-dependent but
independent of the type of nodule formed by the host legume. Nops are beneficial in the symbiosis
with Glycine max and Glycyrrhiza uralensis, and detrimental in the case of the tropical legume
Erythrina variegata
Type VI secretion: a beginner's guide
Type VI secretion is a newly described mechanism for protein transport across the cell envelope of Gram-negative bacteria. Components that have been partially characterised include an IcmF homologue, the ATPase ClpV, a regulatory FHA domain protein and the secreted VgrG and Hcp proteins. Type VI secretion is clearly a key virulence factor for some important pathogenic bacteria and has been implicated in the translocation of a potential effector protein into eukaryotic cells by at least one organism (Vibrio cholerae). However, type VI secretion systems (T6SSs) are widespread in nature and not confined to known pathogens. In accordance with the general rule that the expression of protein secretion systems is tightly regulated, expression of type VI secretion is controlled at both transcriptional and post-transcriptional levels
Development of an improved Pseudoalteromonas haloplanktis TAC125 strain for recombinant protein secretion at low temperature
Background: In a previous paper, we reported the accomplishment of a cold gene-expression system for
the recombinant secretion of heterologous proteins in Pseudoalteromonas haloplanktis TAC125. This
system makes use of the psychrophilic α-amylase from P. haloplanktis TAB23 as secretion carrier, and
allows an effective extra-cellular addressing of recombinant proteins. However, Pseudoalteromonales are
reported to secrete a wide range of extra-cellular proteases. This feature works against the efficiency of
the cold-adapted secretion system, because of the proteolytic degradation of recombinant products. The
aim of this study is the construction of a P. haloplanktis TAC125 mutant strain with reduced extra-cellular
proteolytic activity.
Results: P. haloplanktis TAC125 culture medium resulted to contain multiple and heterogeneous
proteases. Since the annotation of the Antarctic bacterium genome highlighted the presence of only one
canonical secretion machinery, namely the Type II secretion pathway (T2SS), we have inactivated this
secretion system by a gene insertion strategy. A mutant strain of P. haloplanktis TAC125 in which the gspE
gene was knocked-out, actually displayed a remarkable reduction of the extra-cellular protease secretion.
Quite interestingly this strain still retained the ability to secrete the psychrophilic amylase as efficiently as
the wild type. Moreover, the decrease in extra-cellular proteolytic activity resulted in a substantial
improvement in the stability of the secreted amylase-β-lactamase chimera.
Conclusion: Here we report a cell engineering approach to the construction of a P. haloplanktis TAC125
strain with reduced extra-cellular protease activity. The improved strain is able to secrete the
psychrophilic α-amylase (the carrier of our recombinant secretion system), while it displays a significant
reduction of protease content in the culture medium. These features make the gspE mutant an improved
host with a remarkable biotechnological potential in recombinant protein secretion at low temperature.
Moreover this work demonstrates that P. haloplanktis TAC125 is a versatile psychrophilic host for
recombinant protein production since it can be easily improved by a directed engineering approach. To
the best of our knowledge, this is the first described example of a strain improvement strategy applied to
an Antarctic bacterium
Functional analysis of the magnetosome island in Magnetospirillum gryphiswaldense: the mamAB operon is sufficient for magnetite biomineralization
Bacterial magnetosomes are membrane-enveloped, nanometer-sized crystals of magnetite, which serve for magnetotactic navigation. All genes implicated in the synthesis of these organelles are located in a conserved genomic magnetosome island (MAI). We performed a comprehensive bioinformatic, proteomic and genetic analysis of the MAI in Magnetospirillum gryphiswaldense. By the construction of large deletion mutants we demonstrate that the entire region is dispensable for growth, and the majority of MAI genes have no detectable function in magnetosome formation and could be eliminated without any effect. Only <25% of the region comprising four major operons could be associated with magnetite biomineralization, which correlated with high expression of these genes and their conservation among magnetotactic bacteria. Whereas only deletion of the mamAB operon resulted in the complete loss of magnetic particles, deletion of the conserved mms6, mamGFDC, and mamXY operons led to severe defects in morphology, size and organization of magnetite crystals. However, strains in which these operons were eliminated together retained the ability to synthesize small irregular crystallites, and weakly aligned in magnetic fields. This demonstrates that whereas the mamGFDC, mms6 and mamXY operons have crucial and partially overlapping functions for the formation of functional magnetosomes, the mamAB operon is the only region of the MAI, which is necessary and sufficient for magnetite biomineralization. Our data further reduce the known minimal gene set required for magnetosome formation and will be useful for future genome engineering approaches
Use of the KlADH3 promoter for the quantitative production of the murine PDE5A isoforms in the yeast Kluyveromyces lactis
Background: Phosphodiesterases (PDE) are a superfamily of enzymes that hydrolyse cyclic nucleotides (cAMP/
cGMP), signal molecules in transduction pathways regulating crucial aspects of cell life. PDEs regulate the intensity
and duration of the cyclic nucleotides signal modulating the downstream biological efect. Due to this critical role
associated with the extensive distribution and multiplicity of isozymes, the 11 mammalian families (PDE1 to PDE11)
constitute key therapeutic targets. PDE5, one of these cGMP-specifc hydrolysing families, is the molecular target of
several well known drugs used to treat erectile dysfunction and pulmonary hypertension. Kluyveromyces lactis, one of
the few yeasts capable of utilizing lactose, is an attractive host alternative to Saccharomyces cerevisiae for heterologous
protein production. Here we established K. lactis as a powerful host for the quantitative production of the murine
PDE5 isoforms.
Results: Using the promoter of the highly expressed KlADH3 gene, multicopy plasmids were engineered to produce
the native and recombinant Mus musculus PDE5 in K. lactis. Yeast cells produced large amounts of the purifed A1, A2
and A3 isoforms displaying Km, Vmax and Sildenafl inhibition values similar to those of the native murine enzymes.
PDE5 whose yield was nearly 1 mg/g wet weight biomass for all three isozymes (30 mg/L culture), is well tolerated by
K. lactis cells without major growth defciencies and interferences with the endogenous cAMP/cGMP signal transduction
pathways.
Conclusions: To our knowledge, this is the frst time that the entire PDE5 isozymes family containing both regulatory
and catalytic domains has been produced at high levels in a heterologous eukaryotic organism. K. lactis has been
shown to be a very promising host platform for large scale production of mammalian PDEs for biochemical and structural
studies and for the development of new specifc PDE inhibitors for therapeutic applications in many pathologies
Integrated functions among multiple starch synthases determine both amylopectin chain length and branch linkage location in Arabidopsis leaf starch
This study assessed the impact on starch metabolism in Arabidopsis leaves of simultaneously eliminating multiple soluble starch synthases (SS) from among SS1, SS2, and SS3. Double mutant ss1- ss2- or ss1- ss3- lines were generated using confirmed null mutations. These were compared to the wild type, each single mutant, and ss1- ss2- ss3- triple mutant lines grown in standardized environments. Double mutant plants developed similarly to the wild type, although they accumulated less leaf starch in both short-day and long-day diurnal cycles. Despite the reduced levels in the double mutants, lines containing only SS2 and SS4, or SS3 and SS4, are able to produce substantial amounts of starch granules. In both double mutants the residual starch was structurally modified including higher ratios of amylose:amylopectin, altered glucan chain length distribution within amylopectin, abnormal granule morphology, and altered placement of α(1→6) branch linkages relative to the reducing end of each linear chain. The data demonstrate that SS activity affects not only chain elongation but also the net result of branch placement accomplished by the balanced activities of starch branching enzymes and starch debranching enzymes. SS3 was shown partially to overlap in function with SS1 for the generation of short glucan chains within amylopectin. Compensatory functions that, in some instances, allow continued residual starch production in the absence of specific SS classes were identified, probaby accomplished by the granule bound starch synthase GBSS1.ANR Génoplante GPLA0611GEuropean Union-FEDER, Région Nord Pas de Calais ARCir PlantTEQ5National Science Foundation DBI-0209789Comisión Interministerial de Ciencia y Tecnología BIO2009-07040Junta de Andalucía P09-CVI-470
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