Gene Expression In Gram-negative Bacteria Research Articles

Regulation of Acyl Homoserine Lactone Synthesis in Pseudomonas putida JMQS1 Under Phenol Stress

The cell density-dependent gene expression in gram-negative bacteria is through the activity of acyl homoserine lactone signal molecules. The novel strain Pseudomonas putida JMQS1 isolated from detergent-contaminated soil exhibited quorum sensing along with its ability to degrade phenol. The response to Chromobacterium violaceum DSTS-1 mutant biosensor and luxI and luxR gene-specific amplification was used to characterize the quorum sensing property of the isolate. A regulation in the synthesis of various acyl homoserine lactone molecules, viz C6HSL in the initial stages of phenol stress, C8HSL during degradation, and 3OC12HSL on completion of degradation could be identified by liquid chromatography-quadrupole time of flight. Thin-layer chromatography, Fourier transform infrared spectroscopy, and gas chromatography mass spectrometry confirmed the complete degradation of phenol in 48–56 h. P. putida JMQS1 exhibited adaptation over phenol stress through the selective activation of the quorum sensing signal molecules depending on the changing physiological conditions. This study proposes an efficient method for enhancing the degradation of toxic organic pollutants by the supplementation of acyl homoserine lactone signal molecules.

Data on the standardization of a cyclohexanone-responsive expression system for Gram-negative bacteria.

Engineering of robust microbial cell factories requires the use of dedicated genetic tools somewhat different from those traditionally used for laboratory-adapted microorganisms. We have edited and formatted the ChnR/PchnB regulatory node of Acinetobacter johnsonii to ease the targeted engineering of ectopic gene expression in Gram-negative bacteria. The proposed compositional standard was thoroughly verified with a monomeric and superfolder green fluorescent protein (msf•GFP) in Escherichia coli. The expression data presented reflect a tightly controlled transcription initiation signal in response to cyclohexanone. Data in this article are related to the research paper “Genetic programming of catalytic Pseudomonas putida biofilms for boosting biodegradation of haloalkanes” [1].

Relative Strengths of Promoters Provided by Common Mobile Genetic Elements Associated with Resistance Gene Expression in Gram-Negative Bacteria.

Comparison of green fluorescent protein expression from outward-facing promoters (POUT) of ISAba1, ISEcp1, and ISAba125 revealed approximate equivalence in strength, intermediate between PCS (strong) and PCWTGN-10 (weak) class 1 integron promoter variants, >30-fold stronger than POUT of ISCR1, and >5 times stronger than Ptac. Consistent with its usual role, PCWTGN-10 produces more mRNA from a "downstream" gfp gene transcriptionally linked to a "usual" PCWTGN-10-associated gene cassette than does POUT of ISAba1.

Protein oligomers studied by solid‐state NMR – the case of the full‐length nucleoid‐associated protein histone‐like nucleoid structuring protein

Members of the histone-like nucleoid structuring protein (H-NS) family play roles both as architectural proteins and as modulators of gene expression in Gram-negative bacteria. The H-NS protein participates in modulatory processes that respond to environmental changes in osmolarity, pH, or temperature. H-NS oligomerization is essential for its activity. Structural models of different truncated forms are available. However, high-resolution structural details of full-length H-NS and its DNA-bound state have largely remained elusive. We report on progress in characterizing the biologically active H-NS oligomers with solid-state NMR. We compared uniformly ((13)C,(15)N)-labeled ssNMR preparations of the isolated N-terminal region (H-NS 1-47) and full-length H-NS (H-NS 1-137). In both cases, we obtained ssNMR spectra of good quality and characteristic of well-folded proteins. Analysis of the results of 2D and 3D (13)C-(13)C and (15)N-(13)C correlation experiments conducted at high magnetic field led to assignments of residues located in different topological regions of the free full-length H-NS. These findings confirm that the structure of the N-terminal dimerization domain is conserved in the oligomeric full-length protein. Small changes in the dimerization interface suggested by localized chemical shift variations between solution and solid-state spectra may be relevant for DNA recoginition.

Construction of a Broad-Host-Range Tn 7 -Based Vector for Single-Copy P BAD -Controlled Gene Expression in Gram-Negative Bacteria

We describe a mini-Tn7-based broad-host-range expression cassette for arabinose-inducible gene expression from the P(BAD) promoter. This delivery vector, pTJ1, can integrate a single copy of a gene into the chromosome of Gram-negative bacteria for diverse genetic applications, of which several are discussed, using Pseudomonas aeruginosa as the model host.

RelA protein stimulates the activity of RyhB small RNA by acting on RNA-binding protein Hfq

The conserved RNA-binding protein Hfq and its associated small regulatory RNAs (sRNAs) are increasingly recognized as the players of a large network of posttranscriptional control of gene expression in Gram-negative bacteria. The role of Hfq in this network is to facilitate base pairing between sRNAs and their trans-encoded target mRNAs. Although the number of known sRNA-mRNA interactions has grown steadily, cellular factors that influence Hfq, the mediator of these interactions, have remained unknown. We report that RelA, a protein long known as the central regulator of the bacterial-stringent response, acts on Hfq and thereby affects the physiological activity of RyhB sRNA as a regulator of iron homeostasis. RyhB requires RelA in vivo to arrest growth during iron depletion and to down-regulate a subset of its target mRNAs (fdoG, nuoA, and sodA), whereas the sodB and sdhC targets are barely affected by RelA. In vitro studies with recombinant proteins show that RelA enhances multimerization of Hfq monomers and stimulates Hfq binding of RyhB and other sRNAs. Hfq from polysomes extracted from wild-type cells binds RyhB in vitro, whereas Hfq from polysomes of a relA mutant strain shows no binding. We propose that, by increasing the level of the hexameric form of Hfq, RelA enables binding of RNAs whose affinity for Hfq is low. Our results suggest that, under specific conditions and/or environments, Hfq concentrations are limiting for RNA binding, which thereby provides an opportunity for cellular proteins such as RelA to impact sRNA-mediated responses by modulating the activity of Hfq.

The on–off switch of CRISPR immunity against phages in Escherichia coli

The on–off switch of CRISPR immunity against phages in <i>Escherichia coli</i>

A global modulatory role for the Yersinia enterocolitica H-NS protein

The H-NS protein plays a significant role in the modulation of gene expression in Gram-negative bacteria. Whereas isolation and characterization of hns mutants in Escherichia coli, Salmonella and Shigella represented critical steps to gain insight into the modulatory role of H-NS, it has hitherto not been possible to isolate hns mutants in Yersinia. The hns mutation is considered to be deleterious in this genus. To study the modulatory role of H-NS in Yersinia we circumvented hns lethality by expressing in Y. enterocolitica a truncated H-NS protein known to exhibit anti-H-NS activity in E. coli (H-NST(EPEC)). Y. enterocolitica cells expressing H-NST(EPEC) showed an altered growth rate and several differences in the protein expression pattern, including the ProV protein, which is modulated by H-NS in other enteric bacteria. To further confirm that H-NST(EPEC) expression in Yersinia can be used to demonstrate H-NS-dependent regulation in this genus, we used this approach to show that H-NS modulates expression of the YmoA protein.

A family of promoter probe vectors incorporating autofluorescent and chromogenic reporter proteins for studying gene expression in Gram-negative bacteria

A series of promoter probe vectors for use in Gram-negative bacteria has been made in two broad-host-range vectors, pOT (pBBR replicon) and pJP2 (incP replicon). Reporter fusions can be made to gfpUV, gfpmut3.1, unstable gfpmut3.1 variants (LAA, LVA, AAV and ASV), gfp+, dsRed2, dsRedT.3, dsRedT.4, mRFP1, gusA or lacZ. The two vector families, pOT and pJP2, are compatible with one another and share the same polylinker for facile interchange of promoter regions. Vectors based on pJP2 have the advantage of being ultra-stable in the environment due to the presence of the parABCDE genes. As a confirmation of their usefulness, the dicarboxylic acid transport system promoter (dctA(p)) was cloned into a pOT (pRU1097)- and a pJP2 (pRU1156)-based vector and shown to be expressed by Rhizobium leguminosarum in infection threads of vetch. This indicates the presence of dicarboxylates at the earliest stages of nodule formation.

Functional Replacement of the Oligomerization Domain of H-NS by the Hha Protein of Escherichia coli

Members of the H-NS family of proteins play a relevant role as modulators of gene expression in gram-negative bacteria. Interaction of these proteins with members of the Hha/YmoA family of proteins has been previously reported. It has been hypothesized that the latter proteins are functionally equivalent to the N-terminal domain of H-NS-like proteins. In this report we test this assumption by replacing the N-terminal domain of Escherichia coli H-NS by Hha. It has been possible to obtain a functional protein that can compensate for some of the hns-induced phenotypes. These results highlight the relevance of H-NS-Hha interactions to generate heterooligomeric complexes that modulate gene expression in gram-negative bacteria.

Crystal Structure of the N-terminal Dimerisation Domain of VicH, the H-NS-like Protein of Vibrio cholerae

The histone-like nucleoid structuring (H-NS) protein is a global modulator of gene expression in Gram-negative bacteria. VicH, the H-NS protein of Vibrio cholerae, regulates the expression of certain major virulence determinants implicated in the pathogenesis of cholera. We present here the 2.5Å crystal structure of the N-terminal oligomerisation domain of VicH (VicH_Nt). VicH_Nt adopts the same fold and dimeric assembly as the NMR structure of Escherichia coli H-NS_Nt, thus validating this fold against conflicting data. The structural similarity of V.cholerae VicH_Nt and E.coli H-NS_Nt, despite differences in origin, system of expression, experimental conditions and techniques used, indicates that the fold determined in our studies is robust to experimental conditions. Structural analysis and homology modelling were carried out to further elucidate the molecular basis of the functional polyvalence of the N-terminal domain. Our analysis of members of the H-NS superfamily supports the suggestion that the oligomerisation function of H-NS_Nt is conserved even in more distantly related proteins.

Tight regulation and modulation via a C1-regulated promoter in Escherichia coli and Pseudomonas aeruginosa.

We describe the development and analysis of a novel promoter system regulated by the bacteriophage P1 temperature-sensitive C1 repressor. Using transcriptional fusions to the lacZ reporter gene to monitor gene expression, we show that the ratio of induction/repression can be up to 1500-fold in Escherichia coli. The promoters exhibited extremely tight repression and could be modulated over a range of temperatures. The utility of the promoter system was tested in Pseudomonas aeruginosa. C1 effectively repressed transcription; however, only modest induction was achieved. To increase the levels of induction, the amount of c1 was modulated at the mRNA level by using a LacI-regulated promoter. This resulted in a 59-fold induction in gene expression under inducing conditions. As the promoter system was constructed in a broad-host range vector and utilized the C1 repressor from a broad-host range phage, the system will provide the potential for controlled gene expression in Gram-negative bacteria.

Construction of multipurpose gene cartridges based on a novel synthetic promoter for high-level gene expression in gram-negative bacteria.

A series of gene cartridges containing a novel synthetic promoter (Psyn) was constructed. The Psyn sequence is based on the consensus of a number of naturally occurring promoters and displays strong activity in Escherichia coli and Rhizobium leguminosarum. In a direct comparison, Psyn proved to be about twice as strong as the tac promoter in E. coli, while the difference in Rhizobium was about tenfold. A small Psyn cartridge was constructed by adding a Shine-Dalgarno sequence, an ATG codon, and a removable lac operator, whose excision can convert the regulated cartridge into a constitutively expressed unit. A second cassette was obtained by the addition of a lacIq gene in order to provide autonomous regulation also in hosts lacking lacI functions, such as R. leguminosarum. A promoterless lacZ gene was inserted to monitor the activity. This gene can be either replaced with genes of interest, or used for gene fusions by means of conveniently positioned restriction sites. A third cassette was generated by adding a mercury-resistance determinant as a selectable marker, suitable for monitoring tagged bacteria released into environments. In such cases, where a non-antibiotic-resistant marker is preferable, the use of mercury chloride adds the advantage of inhibiting fungal growth when plating soil suspensions. The presence of the second marker, lacZ driven by the strong Psyn, facilitates the selection. Furthermore, the Psyn fragment can be used as a specific probe for the detection of released bacteria engineered with any of the above constructs.

Gene expression using Gram-negative bacteria

The technology for expressing heterologous gene products has developed to the point where it may be successfully applied to almost any Gram-negative bacterial system. Yet, when one thinks of gene expression in Gramnegative bacteria, Escherichia coli is the organism that typically first comes to mind. E. coli has been, and continues to be, the workhorse of the gene expression field. No other system has been developed that can not only express a large number of known gene products at levels sufficient for detailed biochemical analysis or product development, but also express undefined coding sequences (open reading frames) in amounts sufficient to determine the identity of the gene product and to characterize its function. Indeed, literally hundreds of papers are published each year in which it has been reported that genes of prokaryotic or eukaryotic origin have been cloned and expressed in E. coil The ability to express this myriad of genes has been aided by the development of numerous expression vector systems which use highly efficient transcriptional and translational regulatory signals to optimize production to levels that may reach 30% of total cellular protein (for a review of expression vector technology see [ 1] ). However, over the last year or two, the focus of improving expression in E, coli has shifted from the optimization of these regulatory signals to the manipulation of the coding sequence itself and the quality of the protein being produced. This review highlights advances made over the last two years in our ability to optimize the expression of heterologous gene products in E. coli with respect to both the quantity and quality (solubility, folding, activity, and homogeneity) of the proteins being produced. In addition, it will spotlight our emerging abilities to express heterologous gene products in other Gram-negative organisms where the aim is not so much to overproduce these proteins but rather to impart a useful new phenotype or function to these organisms.

R-factor gene expression gram-negative bacteria.

Summary: Methods by which the R-factor 1818 was transferred from Escherichia coli K12 to different species of Gram-negative bacteria are described. Possession of 1818 resulted in the production of similar amounts of R-factor specific penicillinase per organism in E. coli, Serratia marcescens, Alkalescens sp. and Aerobacter aerogenes. The Aerobacter strain produced additionally a ‘chromosomal’ penicillinase which had no influence on the level of R-factor enzyme. Two different strains of Proteus mirabilis containing R-factor 1818 produced only about th of this penicillinase activity per bacterium Two further R-factors 7268 and TEM also specifying penicillinase were introduced into theP. mirabilis strains and enzyme activities per bacterium of about th and th, respectively, were obtained compared with those in E. coli. All organisms in cultures of P. mirabilis exhibited properties of R-factor possession and thus R-factor instability cannot explain these results. It would seem that the phenotypic expression of R-factor penicillinase genes is impaired in this species.