Antibiotic resistance gene cassettes derived from the π interposon for use in E. coli and Streptomyces

doi:10.1016/S0378-1119(97)00014-0

Gene

Volume 190, Issue 2, 6 May 1997, Pages 315-317

https://doi.org/10.1016/S0378-1119(97)00014-0 Get rights and content

Abstract

Three antibiotic resistance gene cassettes, derived from the π interposon (Prentki and Krisch (1984) Gene 29, 303–313) were constructed. These cassettes carry different antibiotic resistance genes, conferring resistance to geneticin, hygromycin or viomycin, flanked by short inverted repeats containing transcription and translation termination signals and synthetic polylinkers. These cassettes were designated πaac, πhyg and πvph. Resistance phenotypes conferred by these constructions are selectable in E. coli and Streptomyces. These cassettes can be used for insertional mutagenesis or for vector construction.

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Unidirectional growth of filamentous protein assemblies including the bacterial flagellum relies on dedicated polymerization factors (PFs). The molecular determinants and structural transitions imposed by PFs on multi-subunit assembly are poorly understood. Here, we unveil FlaY from the polarized α-proteobacterium Caulobacter crescentus as a defining member of an alternative class of specialized flagellin PFs. Unlike the paradigmatic FliD capping protein, FlaY relies on a funnel-like β-propeller fold for flagellin polymerization. FlaY binds flagellin and is secreted by the flagellar secretion apparatus, yet it can also promote flagellin polymerization exogenously when donated from flagellin-deficient cells, serving as a transferable, extracellular public good. While the surge in FlaY abundance precedes bulk flagellin synthesis, FlaY-independent filament assembly is enhanced by mutation of a conserved region in multiple flagellin paralogs. We suggest that FlaYs are (multi-)flagellin PFs that evolved convergently to FliDs yet appropriated the versatile β-propeller fold implicated in human diseases for chaperone-assisted filament assembly.
Regulation of the high-specificity Rubisco genes by the third CbbR-type regulator in a hydrogen-oxidizing bacterium Hydrogenovibrio marinus
2022, Journal of Bioscience and Bioengineering
The obligate chemolithoautotrophic bacterium, Hydrogenovibrio marinus MH-110, has three ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) isoenzymes, CbbM, CbbLS-1, and CbbLS-2, which differ in CO₂/O₂ specificity factor values. Expressions of CbbM and CbbLS-1 are regulated differently by transcriptional regulators of the LysR family, CbbRm and CbbR1, respectively. CbbLS-2 has the highest specificity and is induced under low CO₂ conditions, but the regulator for the cbbL2S2 genes encoding CbbLS-2 remains unidentified. In this study, the cbbR2 gene encoding the third CbbR-type regulator was identified in the downstream region of the cbbL2S2 and carboxysome gene cluster via transposon mutagenesis. CO₂ depletion induced the cbbR2 gene. The cbbR2 knockout mutant could not grow under low CO₂ conditions and did not produce CbbLS-2. Recombinant CbbR2 protein was bound to the promoter region of the cbbL2S2 genes. These results indicate that CbbR2 is the specific regulator for CbbLS-2 expression.
Inner membrane YfgM–PpiD heterodimer acts as a functional unit that associates with the SecY/E/G translocon and promotes protein translocation
2022, Journal of Biological Chemistry
PpiD and YfgM are inner membrane proteins that are both composed of an N-terminal transmembrane segment and a C-terminal periplasmic domain. Escherichia coli YfgM and PpiD form a stable complex that interacts with the SecY/E/G (Sec) translocon, a channel that allows protein translocation across the cytoplasmic membrane. Although PpiD is known to function in protein translocation, the functional significance of PpiD–YfgM complex formation as well as the molecular mechanisms of PpiD–YfgM and PpiD/YfgM–Sec translocon interactions remain unclear. Here, we conducted genetic and biochemical studies using yfgM and ppiD mutants and demonstrated that a lack of YfgM caused partial PpiD degradation at its C-terminal region and hindered the membrane translocation of Vibrio protein export monitoring polypeptide (VemP), a Vibrio secretory protein, in both E. coli and Vibrio alginolyticus. While ppiD disruption also impaired VemP translocation, we found that the yfgM and ppiD double deletion exhibited no additive or synergistic effects. Together, these results strongly suggest that both PpiD and YfgM are required for efficient VemP translocation. Furthermore, our site-directed in vivo photocrosslinking analysis revealed that the tetratricopeptide repeat domain of YfgM and a conserved structural domain (NC domain) in PpiD interact with each other and that YfgM, like PpiD, directly interacts with the SecG translocon subunit. Crosslinking analysis also suggested that PpiD–YfgM complex formation is required for these proteins to interact with SecG. In summary, we propose that PpiD and YfgM form a functional unit that stimulates protein translocation by facilitating their proper interactions with the Sec translocon.
Caulobacter lipid A is conditionally dispensable in the absence of fur and in the presence of anionic sphingolipids
2022, Cell Reports
Lipid A, the membrane-anchored portion of lipopolysaccharide (LPS), is an essential component of the outer membrane (OM) of nearly all Gram-negative bacteria. Here we identify regulatory and structural factors that together render lipid A nonessential in Caulobacter crescentus. Mutations in the ferric uptake regulator fur allow Caulobacter to survive in the absence of either LpxC, which catalyzes an early step of lipid A synthesis, or CtpA, a tyrosine phosphatase homolog we find is needed for wild-type lipid A structure and abundance. Alterations in Fur-regulated processes, rather than iron status per se, underlie the ability to survive when lipid A synthesis is blocked. Fitness of lipid A-deficient Caulobacter requires an anionic sphingolipid, ceramide phosphoglycerate (CPG), which also mediates sensitivity to the antibiotic colistin. Our results demonstrate that, in an altered regulatory landscape, anionic sphingolipids can support the integrity of a lipid A-deficient OM.
Regulation of aureofuscin production by the PAS-LuxR family regulator AurJ3M
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aurJ3M(GenBank:EU697915.1), a 579 bp gene, whose deduced product (192 amino acid) was found to have amino acid sequence homology with some bacterial regulatory proteins. Computer-assisted analysis showed that AurJ3M is PAS-LuxR family regulatory protein, it combines a PAS domain with a helix–turn–helix (HTH) motif of the LuxR type. Gene deletion of aurJ3M from the S.aureofuscus SYAU0709 through gene replacement with the apramycin resistance gene aac (3) IV and the DNA fragment of the replication initiation site sequence OriT resulted in complete loss of aureofuscin production, indicating that AurJ3M is a positive regulator during the aureofuscin biosynthesis.
Gene expression analyses in S.aureofuscus SYAU0709 and S.aureofuscus ΔaurJ3M by reverse transcriptase PCR (RT-PCR) indicated there no transcripts for the genes B、C、G、F、S0、S1、D in S.aureofuscus ΔaurJ3M, maintains some transcription of these genes although the levels is low. Transcription was also significantly reduced for gene A, and no difference in the transcription pattern was observed for the genes R、E and H, a similar transcription pattern was also observed for AurJ3M, A and B showed a different transcription pattern, while transcription of gene A in S.aureofuscus ΔaurJ3M when compared to the parental strain, no transcripts could be detected for gene B in the mutant; This result indicates that the deletion of aurJ3M gene doesn't have a polar effect on the transcription of genes located downstream from aurJ3M. In the ΔaurJ3M knockout mutant, except for the transcription of E、H、M and R, the transcription of other genes in the cluster was down-regulated, HPLC shows there no secondary metabolites was produced,aurJ3M has a pathway-specific regulation effect on the biosynthesis of aureofuscin. Aureofuscin had broad application prospects in food preservation, and enriched the resource pool of anti-fungal antibiotics of tetraphenyl macrolide in China.
Function of three RuBisCO enzymes under different CO<inf>2</inf> conditions in Hydrogenovibrio marinus
2018, Journal of Bioscience and Bioengineering
The obligate chemolithoautotrophic bacterium, Hydrogenovibrio marinus MH-110 has three ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) isoenzymes, designated CbbLS-1, CbbLS-2, and CbbM, which are encoded by the cbbL1S1, cbbL2S2, and cbbM genes, respectively. Functions of these isoenzymes at different CO₂ concentrations were investigated using deletion mutants of their genes. Deletion of cbbL1 had no effect on cell growth under any of the test growth conditions. The cbbL2 mutant was unable to grow under lower (≤0.15%) CO₂ conditions, though it grew normally under higher (≥2%) CO₂ conditions. Growth of the cbbM mutant was retarded under higher CO₂ conditions but was not affected by lower CO₂ conditions. These results indicate that CbbLS-2 and CbbM specifically function under lower and higher CO₂ conditions, respectively. The growth retardation of the cbbL2 and cbbM mutants was not restored by complementation with plasmids carrying the cbbL2S2 and cbbM genes, respectively. The cbbL2S2 and cbbM genes are followed by the carboxysome genes and the cbbQmOm genes, respectively. Co-expression of these downstream genes was probably necessary for the in vivo function of CbbLS-2 and CbbM. CbbLS-1 was upregulated in the cbbL2 and cbbM mutants under the lower and higher CO₂ conditions, respectively, indicating that the expression of cbbL1S1 was controlled to compensate the deficiency of the other RuBisCO isoenzymes.

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^☆: On request, the authors will supply detailed experimental evidence for the conclusions reached in this Short Communication.

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Abstract

References (12)

Interposon mutagenesis of soil and water bacteria: a family of DNA fragments designed for in vitro insertional mutagenesis of Gram-negative bacteria

Gene

In vitro insertional mutagenesis with a selectable DNA fragment

Gene

Plasmid vectors for selecting ISl-promoted deletions in cloned DNA-sequence analysis of the Omega interposon

Gene

A new shuttle cosmid vector, pKC505, for streptomycetes: its use in the cloning of three different spiramycin-resistance genes from a Streptomyces ambofaciens library

Gene

Genes for gentamicin-(3)-N-acetyl-transferases III and IV: II

Nucleotide sequences encoding and promoting expression of three antibiotic resistance genes indigenous to Streptomyces

Mol. Gen. Genet.

Cited by (186)

Extracellular transfer of a conserved polymerization factor for multi-flagellin filament assembly in Caulobacter

Regulation of the high-specificity Rubisco genes by the third CbbR-type regulator in a hydrogen-oxidizing bacterium Hydrogenovibrio marinus

Inner membrane YfgM–PpiD heterodimer acts as a functional unit that associates with the SecY/E/G translocon and promotes protein translocation

Caulobacter lipid A is conditionally dispensable in the absence of fur and in the presence of anionic sphingolipids

Regulation of aureofuscin production by the PAS-LuxR family regulator AurJ3M

Function of three RuBisCO enzymes under different CO<inf>2</inf> conditions in Hydrogenovibrio marinus

Short communicationAntibiotic resistance gene cassettes derived from the π interposon for use in E. coli and Streptomyces☆

Abstract

Gene

Gene

Gene

Gene

Genes for gentamicin-(3)-N-acetyl-transferases III and IV: II

Nucleotide sequences encoding and promoting expression of three antibiotic resistance genes indigenous to Streptomyces

Mol. Gen. Genet.

Short communication
Antibiotic resistance gene cassettes derived from the π interposon for use in E. coli and Streptomyces☆