Construction of a Xylanase-Producing Strain of
 Brevibacterium
 lactofermentum
 by Stable Integration of an Engineered
 xysA
 Gene from
 Streptomyces
 halstedii
 JM8

Adham, Sirin A.; Campelo, Ana B.; Ramos, Alberto; Gil, José A.

doi:10.1128/aem.67.12.5425-5430.2001

Cited by 20 publications

(8 citation statements)

References 22 publications

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“…The exogenous genes were cloned behind the divIVA Cg promoter (Pdiv), and the construct was inserted as a single copy by homologous recombination into the region upstream from divIVA in the C. glutamicum GNTD chromosome ( Fig. 2A), as previously described for other genes (1). Similar constructs carrying translational fusions between divIVA genes and egfp2 were also produced and integrated into strain GNTD (Fig.…”

Section: Resultsmentioning

confidence: 99%

DivIVA Is Required for Polar Growth in the MreB-Lacking Rod-Shaped ActinomyceteCorynebacterium glutamicum

et al. 2008

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The actinomycete Corynebacterium glutamicum grows as rod-shaped cells by zonal peptidoglycan synthesis at the cell poles. In this bacterium, experimental depletion of the polar DivIVA protein (DivIVA Cg ) resulted in the inhibition of polar growth; consequently, these cells exhibited a coccoid morphology. This result demonstrated that DivIVA is required for cell elongation and the acquisition of a rod shape. DivIVA from Streptomyces or Mycobacterium localized to the cell poles of DivIVA Cg -depleted C. glutamicum and restored polar peptidoglycan synthesis, in contrast to DivIVA proteins from Bacillus subtilis or Streptococcus pneumoniae, which localized at the septum of C. glutamicum. This confirmed that DivIVAs from actinomycetes are involved in polarized cell growth. DivIVA Cg localized at the septum after cell wall synthesis had started and the nucleoids had already segregated, suggesting that in C. glutamicum DivIVA is not involved in cell division or chromosome segregation.

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Section: Resultsmentioning

confidence: 99%

DivIVA Is Required for Polar Growth in the MreB-Lacking Rod-Shaped ActinomyceteCorynebacterium glutamicum

et al. 2008

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“…1), plasmid pK18-3DZ (Table 1) was introduced by conjugation into C. glutamicum to insert the partial ftsZ gene in a non-essential chromosomal region using a strategy designed to introduce any gene into the C. glutamicum chromosome (Adham et al, 2001a). The new strain, C. glutamicum AR12, contained the partial ftsZ and the original ftsZ under the control of their endogenous promoters (Fig.…”

Section: Construction Of C Glutamicum Strains Carrying a Unique Compmentioning

confidence: 99%

Altered morphology produced by ftsZ expression in Corynebacterium glutamicum ATCC 13869

et al. 2005

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Corynebacterium glutamicum is a Gram-positive bacterium that lacks the cell division FtsA protein and actin-like MreB proteins responsible for determining cylindrical cell shape. When the cell division ftsZ gene from C. glutamicum (ftsZ Cg ) was cloned in different multicopy plasmids, the resulting constructions could not be introduced into C. glutamicum; it was assumed that elevated levels of FtsZ Cg result in lethality. The presence of a truncated ftsZ Cg and a complete ftsZ Cg under the control of Plac led to a fourfold reduction in the intracellular levels of FtsZ, generating aberrant cells displaying buds, branches and knots, but no filaments. A 20-fold reduction of the FtsZ level by transformation with a plasmid carrying the Escherichia coli lacI gene dramatically reduced the growth rate of C. glutamicum, and the cells were larger and club-shaped. Immunofluorescence microscopy of FtsZ Cg or visualization of FtsZ Cg -GFP in C. glutamicum revealed that most cells showed one fluorescent band, most likely a ring, at the mid-cell, and some cells showed two fluorescent bands (septa of future daughter cells). When FtsZ Cg -GFP was expressed from Plac, FtsZ rings at mid-cell, or spirals, were also clearly visible in the aberrant cells; however, this morphology was not entirely due to GFP but also to the reduced levels of FtsZ expressed from Plac. Localization of FtsZ at the septum is not negatively regulated by the nucleoid, and therefore the well-known occlusion mechanism seems not to operate in C. glutamicum.

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“…The resulting fragment was digested with NdeI and XhoI and ligated into plasmid pXHis1 [28] (Table 2) digested with the same enzymes to obtain plasmid pXHis1-Anti, which was used as an intermediate plasmid. Plasmid pN702Gem3-Anti was obtained by digesting pXHis1-Anti with BglII, purifying the corresponding yefMsl band and ligating it into pN702Gem3 [29] digested with the same enzyme.…”

Section: Methodsmentioning

confidence: 99%

Stable expression plasmids for Streptomyces based on a toxin-antitoxin system

2013

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BackgroundBacteria included in the genus Streptomyces exhibit several attractive characteristics that make them adequate hosts for the heterologous expression of proteins. One of them is that some of its species have a high secretion capacity and hence the protein of interest could be released to the culture supernatant, facilitating downstream processing. To date, all the expression vectors described for these bacteria contain antibiotic resistance genes as selection markers. However, the use of antibiotics to produce proteins at industrial level is currently becoming more restricted owing to the possibility of contamination of the final product. In this report, we describe the use of the S. lividans yefM/yoeBsl toxin-antitoxin system to develop a stable plasmid expression system.ResultsIn order to use the yefM/yoeBsl system to stabilize expression plasmids in Streptomyces, a S. lividans mutant strain that contained only the toxin gene (yoeBsl) in its genome and the antitoxin gene (yefMsl) located in a temperature-sensitive plasmid was constructed and used as host. This strain was transformed with an expression plasmid harbouring both the antitoxin gene and the gene encoding the protein of interest. Thus, after elimination of the temperature-sensitive plasmid, only cells with the expression plasmid were able to survive. On using this system, two proteins - an α-amylase from S. griseus and a xylanase from S. halstedii - were overproduced without the addition of antibiotic to the culture medium. The production of both proteins was high, even after long incubations (8 days), and after serial subcultures, confirming the stability of the plasmids without antibiotic selection.ConclusionsThis is the first report that describes the use of a toxin-antitoxin system to maintain high -copy plasmids in Streptomyces. This finding could be a valuable tool for using Streptomyces as a host to produce proteins at the industrial and pharmaceutical levels without the use of antibiotics in the production step.

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Construction of a Xylanase-Producing Strain of Brevibacterium lactofermentum by Stable Integration of an Engineered xysA Gene from Streptomyces halstedii JM8

Cited by 20 publications

References 22 publications

DivIVA Is Required for Polar Growth in the MreB-Lacking Rod-Shaped ActinomyceteCorynebacterium glutamicum

DivIVA Is Required for Polar Growth in the MreB-Lacking Rod-Shaped ActinomyceteCorynebacterium glutamicum

Altered morphology produced by ftsZ expression in Corynebacterium glutamicum ATCC 13869

Stable expression plasmids for Streptomyces based on a toxin-antitoxin system

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