Chloramphenicol Resistance Cassette Research Articles

An updated system for the targeted mutagenesis of the psbDI:psbC operon in Synechocystis sp. PCC 6803: mutations targeting Asp460 in CP43 of Photosystem II reduce oxygen-evolving activity and perturb electron transfer in the quinone-Fe-acceptor complex

ABSTRACT The cyanobacterium Synechocystis sp. PCC 6803 is widely used as a model organism to study Photosystem II (PS II) of oxygenic photosynthesis. In response to inherent genetic instability in cyanobacteria we re-sequenced our strain of Synechocystis sp. PCC 6803, designated as Glucose Tolerant Otago 1 (GT-O1), to establish its genomic background [Morris JN, Crawford TS, Jeffs A, Stockwell PA, Eaton-Rye JJ, Summerfield TC. 2014. New Zealand Journal of Botany. 52:36–47]. Here we have constructed a mutagenesis system to enable us to study roles of the D2 and CP43 PS II reaction centre proteins in the GT-O1 strain. A ΔDIC/ΔDII deletion mutant was assembled in which the psbDI/psbC operon encoding D2 and CP43 was replaced by a chloramphenicol-resistance cassette and psbDII, encoding a second copy of D2, was replaced with a kanamycin-resistance cassette. A DIC+/DII– control strain was constructed by reintroducing the psbDI/psbC operon under selection provided by a spectinomycin-resistance cassette. In this system mutations can be introduced following in vitro mutagenesis of the psbDI/psbC operon. To test our system, we introduced mutations at the CP43 residue Asp460 which is hydrogen bonded to Arg-24 and Arg-28 of the PsbT low-molecular-weight subunit of PS II that is found at the monomer–monomer interface of the PS II dimeric complex in the thylakoid membrane. While the control strain showed a similar phenotype to wild type our CP43 mutants (D460A, D460E and D460N) exhibited a reduction of oxygen-evolving activity and alteration in electron transfer between the primary (QA) and secondary (QB) plastoquinone electron acceptors of PS II.

The IBD candidate gene, PTPN2, Restricts Epithelial and Macrophage Invasion of a Novel Reporter Adherent‐Invasive Escherichia coli

BackgroundDysbiosis of the gut microbiota and expansion of pathobionts such as adherent‐invasive Escherichia coli (AIEC) are associated with the pathogenesis of Inflammatory Bowel Disease (IBD). Mice deficient in expression of the IBD gene candidate, Ptpn2, exhibit pronounced expansion of a novel murine adherent‐invasive Escherichia coli (mAIEC). The aim of this study was to generate a fluorescent mAIEC to study host susceptibility to mAIEC invasion, and to identify how loss of PTPN2 expression in host cells modifies mAIEC invasiveness.MethodsmAIEC was isolated from the distal colon of a Ptpn2 knockout (KO) mouse. mAIEC was made chemically competent and then transformed with a plasmid expressing mCherry fluorescent protein and a chloramphenicol resistance cassette. For bacterial adherence and invasion studies, parental Caco‐2bbe cells were infected with mAIEC‐mCherry at multiplicity of infection (MOI) of 10 for 3 hours. Caco‐2bbe intestinal epithelial cells (IEC), stably expressing either scrambled shRNA or shRNA against Ptpn2 were infected with mAIEC‐mCherry (MOI=10) for 5 hours. Peritoneal macrophages from Ptpn2 wildtype, heterozygous and KO mice were infected with mAIEC‐mCherry for 5 hours.ResultsWe previously demonstrated that mAIEC can adhere to and invade IECs. We next validated that mAIEC‐mCherry adhered to (0.94 ±0.61 bacteria/cell compared to control E. coli K12; 0.14±0.03 bacteria/cell, n=4, P=0.0039) IECs. mAIEC‐mCherry had comparable adherence (n=4, P=0.35) to IECs as control (unlabeled) mAIEC. Bacterial invasion of mAIEC‐mCherry invasion of IECs was visually confirmed by fluorescence microscopy. To better understand if the loss of expression of PTPN2, which drives increased luminal abundance of mAIEC, also affected mAIEC pathogenicity, we next tested if Ptpn2 deficiency in host cells altered mAIEC‐mCherry invasion. We observed by immunofluorescence that Ptpn2 knockdown (KD) IECs were more susceptible to mAIEC‐mCherry invasion compared to control IECs (n=3). We also confirmed that macrophages from Ptpn2‐KO mice were more susceptible to mAIEC‐mCherry invasion compared to wildtype and Ptpn2‐heterozygous mouse macrophages (n=5, wildtype vs. KO, P=0.0044; KO vs. heterozygous, P=−0.8665). Ptpn2‐heterozygous macrophages were also more susceptible to invasion than wildtype, indicating a gene‐dosing effect of macrophage PTPN2 loss on bacterial invasion (wildtype vs. heterozygous, P=0.0117). These data identify that loss of PTPN2 expression increases susceptibility to mAIEC invasion.ConclusionHere, we validated a newly generated fluorescently tagged mAIEC to study interactions between this novel mouse AIEC with host cells. We further demonstrated that Ptpn2 deficiency promotes mAIEC of intestinal epithelial cells and macrophages. These data suggest that IBD patients with loss‐of‐function mutations in PTPN2 may have increased susceptibility to colonization by disease‐relevant AIEC.Support or Funding InformationNIH 2R01DK091281 (DFM)

Two novel transcriptional reporter systems for monitoring Helicobacter pylori stress responses

Helicobacter pylori, a human pathogen linked to many stomach diseases, is well adapted to colonize aggressive gastric environments, and its virulence factors contribute this adaptation. Here, we report the construction of two novel H. pylori vectors, pSv2 and pSv4, carrying a reporter gene fused to the promoters of virulence factor genes for monitoring the response of single H. pylori cells to various stresses. H. pylori cryptic plasmids were modified by the introduction of the Escherichia coli origin of replication, chloramphenicol resistance cassette, and promoterless gfp gene to produce E. coli/H. pylori shuttle vectors. The promoter regions of vacA and ureA genes encoding well-characterized H. pylori virulence factors were fused to the promoterless gfp gene. Recording the GFP fluorescence signal from the genetically modified H. pylori cells immobilized in specifically designed microfluidic devices revealed the response of transcriptional reporter systems to osmotic stress, acidic stress, elevated Ni2+ concentration or iron chelation. Our observations validate the utility of the pSv2 and pSv4 vectors to monitor the regulation of virulence factor genes in diverse strains and clinical isolates of H. pylori.

An improved system for the targeted mutagenesis of the psbA2 gene in Synechocystis sp. PCC 6803: mutation of D1-Glu244 to His impairs electron transfer between QA and QB of Photosystem II

ABSTRACTThe cyanobacterium Synechocystis sp. PCC 6803 has three copies of the psbA gene encoding the D1 reaction centre protein of Photosystem II (PS II). We have designed a mutagenesis system that allows introduction of mutations into the constitutively expressed psbA2 copy without affecting the expression of any flanking genes. A triple deletion strain was constructed in which psbA1 and psbA3 were removed by markerless deletions and psbA2 was replaced by a chloramphenicol-resistance cassette. A vector was then designed to enable the reintroduction of psbA2 in the chromosome using a kanamycin-resistance cassette as a selectable marker. This system was used to generate a control strain, which had an unmodified copy of psbA2, and two mutant strains which contained a copy of psbA2 where the codon for D1-Glu244 had been mutated to code for a His or Asp. The D1-Glu244 residue was targeted as it has been hypothesised to participate in a hydrogen-bond network that is required for protonation of the PS II secondary plastoquinone electron acceptor QB. While the phenotype of the control strain was similar to wild type, the E244H mutant exhibited impaired oxygen evolution and altered electron transfer between the primary quinone acceptor QA and QB. However, substitution of Glu-244 by Asp resulted in a mutant more closely resembling the control strain.

Construction of a New Phage Integration Vector pFIV-Val for Use in Different Francisella Species.

We recently identified and described a putative prophage on the genomic island FhaGI-1 located within the genome of Francisella hispaniensis AS02-814 (F. tularensis subsp. novicida-like 3523). In this study, we constructed two variants of a Francisella phage integration vector, called pFIV1-Val and pFIV2-Val (Francisella Integration Vector-tRNAVal-specific), using the attL/R-sites and the site-specific integrase (FN3523_1033) of FhaGI-1, a chloramphenicol resistance cassette and a sacB gene for counter selection of transformants against the vector backbone. We inserted the respective sites and genes into vector pUC57-Kana to allow for propagation in Escherichia coli. The constructs generated a circular episomal form in E. coli which could be used to transform Francisella spp. where FIV-Val stably integrated site specifically into the tRNAVal gene of the genome, whereas pUC57-Kana is lost due to counter selection. Functionality of the new vector was demonstrated by the successfully complementation of a Francisella mutant strain. The vectors were stable in vitro and during host-cell infection without selective pressure. Thus, the vectors can be applied as a further genetic tool in Francisella research, expanding the present genetic tools by an integrative element. This new element is suitable to perform long-term experiments with different Francisella species.

Establishment of a markerless gene deletion system in Chromohalobacter salexigens DSM 3043.

Chromohalobacter salexigens DSM 3043 can grow over a wide range of salinity, which makes it as an excellent model organism for understanding the mechanism of prokaryotic osmoregulation. Functional analysis of C. salexigens genes is an essential way to reveal their roles in cellular osmoregulation. However, the lack of an effective markerless gene deletion system has prevented construction of multiple gene deletion mutants for the members in the genus. Here, we report the development of a markerless gene deletion system in C. salexigens using allelic exchange method. In this system, the in vitro mutant allele of target gene was inserted into a pK18mobsacB-based integrative vector pMDC21, which contained a chloramphenicol resistance cassette as the positive selection marker and a sacB gene from Bacillus subtilis as the counterselectable marker. To validate this system, two single-gene deletion mutants and a double-gene deletion mutant were constructed. In addition, our results showed that growth of the merodiploids and sucrose screening at 25°C were more effective to decrease the occurrence of spontaneous sucrose resistance colonies than at higher temperature (30 or 37°C), and growth of the merodiploids in mineral salt medium instead of the complex medium was critical to increase the recovery rate of deletion mutants.

Identification of a Campylobacter coli methyltransferase targeting adenines at GATC sites.

Campylobacter coli can infect humans and colonize multiple other animals, but its host-associated genes or adaptations are poorly understood. Adenine methylation at GATC sites, resulting in MboI resistance of genomic DNA, was earlier frequently detected among C.coli from swine but not among turkey-derived isolates. The underlying genetic basis has remained unknown. Comparative genome sequence analyses of C.coli 6461, a swine-derived strain with MboI-resistant DNA, revealed two chromosomal ORFs, 0059 and 0060, encoding a putative DNA methyltransferase and a conserved hypothetical protein, respectively, which were lacking from the genome of the turkey-derived C.coli strain 11601, which had MboI-susceptible DNA. To determine whether ORF0059 mediated MboI resistance and hence encoded a putative N6-adenine DNA methyltransferase, the gene was cloned immediately upstream of a chloramphenicol resistance cassette (cat) and a PCR fragment harboring ORF0059-cat was transformed into C.coli 11601. The transformants had MboI-resistant DNA, suggesting a direct role of this gene in methylation of adenines at GATC sites. In silico analyses suggested that the ORF0059-ORF0060 cassette was more frequent among C.coli from swine than certain other sources (e.g. cattle, humans). Potential impacts of ORF0059-mediated methylation on C.coli host preference and other adaptations remain to be elucidated.

Identification of the methyltransferase targeting C2499 in Deinococcus radiodurans 23S ribosomal RNA.

The bacterium Deinococcus radiodurans—like all other organisms—introduces nucleotide modifications into its ribosomal RNA. We have previously found that the bacterium contains a Carbon-5 methylation on cytidine 2499 of its 23S ribosomal RNA, which is so far the only modified version of cytidine 2499 reported. Using homology search, we identified the open reading frame DR_0049 as the primary candidate gene for the methyltransferase that modifies cytidine 2499. Mass spectrometric analysis demonstrated that recombinantly expressed DR0049 protein methylates E. coli cytidine 2499 both in vitro and in vivo. We also inactivated the DR_0049 gene in D. radiodurans through insertion of a chloramphenicol resistance cassette. This resulted in complete absence of the cytidine 2499 methylation, which all together demonstrates that DR_0049 encodes the methyltransferase producing m5C2499 in D. radiodurans 23S rRNA. Growth experiments disclosed that inactivation of DR_0049 is associated with a severe growth defect, but available ribosome structures show that cytidine 2499 is positioned very similar in D. radiodurans harbouring the modification and E. coli without the modification. Hence there is no obvious structure-based explanation for the requirement for the C2499 posttranscriptional modification in D. radiodurans.Electronic supplementary materialThe online version of this article (doi:10.1007/s00792-015-0800-z) contains supplementary material, which is available to authorised users.

Chromosomal integration vectors allowing flexible expression of foreign genes in Campylobacter jejuni.

BackgroundCampylobacter jejuni is a major cause of human gastroenteritis yet there is limited knowledge of how disease is caused. Molecular genetic approaches are vital for research into the virulence mechanisms of this important pathogen. Vectors that allow expression of genes in C. jejuni via recombination onto the chromosome are particularly useful for genetic complementation of insertional knockout mutants and more generally for expression of genes in particular C. jejuni host backgrounds.MethodsA series of three vectors that allow integration of genes onto the C. jejuni chromosome were constructed by standard cloning techniques with expression driven from three different strong promoters. Following integration onto the C. jejuni chromosome expression levels were quantified by fluorescence measurements and cells visualized by fluorescence microscopy.ResultsWe have created plasmid, pCJC1, designed for recombination-mediated delivery of genes onto the C. jejuni chromosome. This plasmid contains a chloramphenicol resistance cassette (cat) with upstream and downstream restriction sites, flanked by regions of the C. jejuni pseudogene Cj0223. Cloning of genes immediately upstream or downstream of the cat gene allows their subsequent introduction onto the C. jejuni chromosome within the pseudogene. Gene expression can be driven from the native gene promoter if included, or alternatively from the cat promoter if the gene is cloned downstream of, and in the same transcriptional orientation as cat. To provide increased and variable expression of genes from the C. jejuni chromosome we modified pCJC1 through incorporation of three relatively strong promoters from the porA, ureI and flaA genes of C. jejuni, Helicobacter pylori and Helicobacter pullorum respectively. These promoters along with their associated ribosome binding sites were cloned upstream of the cat gene on pCJC1 to create plasmids pCJC2, pCJC3 and pCJC4. To test their effectiveness, a green fluorescent protein (gfp) reporter gene was inserted downstream of each of the three promoters and following integration of promoter-gene fusions onto the C. jejuni host chromosome, expression levels were quantified. Expression from the porA promoter produced the highest fluorescence, from flaA intermediate levels and from ureI the lowest. Expression of gfp from the porA promoter enabled visualization by fluorescent microscopy of intracellular C. jejuni cells following invasion of HeLa cells.ConclusionsThe plasmids constructed allow stable chromosomal expression of genes in C. jejuni and, depending on the promoter used, different expression levels were obtained making these plasmids useful tools for genetic complementation and high level expression.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0559-5) contains supplementary material, which is available to authorized users.

Bacilysin overproduction in Bacillus amyloliquefaciens FZB42 markerless derivative strains FZBREP and FZBSPA enhances antibacterial activity.

Bacillus amyloliquefaciens strains FZBREP and FZBSPA were derived from the wild-type FZB42 by replacement of the native bacilysin operon promoter with constitutive promoters P repB and P spac from plasmids pMK3 and pLOSS, respectively. These strains contained two antibiotic resistance genes, and markerless strains were constructed by deleting the chloramphenicol resistance cassette and promoter region bordered by two lox sites (lox71 and lox66) using Cre recombinase expressed from the temperature-sensitive vector pLOSS-cre. The vector-encoded spectinomycin resistance gene was removed by high temperature (50 °C) treatment. RT-PCR and qRT-PCR results indicated that P repB and especially P spac significantly increased expression of the bac operon, and FZBREP and FZBSPA strains produced up to 170.4 and 315.6% more bacilysin than wild type, respectively. Bacilysin overproduction was accompanied by enhancement of the antagonistic activities against Staphylococcus aureus (an indicator of bacilysin) and Clavibacter michiganense subsp. sepedonicum (the causative agent of potato ring rot). Both the size and degree of ring rot-associated necrotic tubers were decreased compared with the wild-type strain, which confirmed the protective effects and biocontrol potential of these genetically engineered strains.

Colonization resistance against genetically modifiedEscherichia coliK12 (W3110) strains is abrogated following broad-spectrum antibiotic treatment and acute ileitis

Escherichia coli K12 (EcK12) is commonly used for gene technology purposes and regarded as a security strain due to its inability to adhere to epithelial cells. The conventional intestinal microbiota composition is critical for physiological colonization resistance against most bacterial species including pathogens. We were therefore interested whether intestinal colonization by a genetically modified EcK12 (W3110) strain carrying a chloramphenicol resistance cassette was facilitated following broad-spectrum antibiotic treatment eradicating the intestinal microbiota or induction of small intestinal inflammation accompanied by distinct microbiota shifts. Whereas conventional C57BL/6 and BALB/c mice had virtually expelled the EcK12 (W3110) strain within the first 3 days upon peroral infection, EcK12 (W3110) could establish within the small and large intestines of gnotobiotic mice generated by quintuple antibiotic treatment. Gnotobiotic mice perorally infected with EcK12 (W3110) plus fecal transplant from conventional donors harbored lower intestinal EcK12 (W3110) loads compared to animals challenged with EcK12 (W3110) alone. Furthermore, EcK12 (W3110) infection of conventional mice after but not before induction of ileitis resulted in stable colonization of ileum and colon by EcK12 (W3110). Taken together, broad-spectrum antibiotic treatment and intestinal inflammation compromise colonization resistance and thus facilitate colonization of the intestinal tract with genetically modified EcK12 security strains.

Insertion mutations in Helicobacter pylori flhA reveal strain differences in RpoN-dependent gene expression

Flagellar biogenesis in the gastric pathogen Helicobacter pylori involves a transcriptional hierarchy that utilizes all three sigma factors found in this bacterium (RpoD, RpoN and FliA). Transcription of the RpoN-dependent genes requires the sensor kinase FlgS and response regulator FlgR. It is thought that FlgS senses some cellular cue to regulate transcription of the RpoN-dependent flagellar genes, but this signal has yet to be identified. Previous studies showed that transcription of the RpoN-dependent genes is inhibited by mutations in flhA, which encodes a membrane-bound component of the flagellar protein export apparatus. We found that depending on the H. pylori strain used, insertion mutations in flhA had different effects on expression of RpoN-dependent genes. Mutations in flhA in H. pylori strains B128 and ATCC 43504 (the type strain) were generated by inserting a chloramphenicol resistance cassette so as to effectively eliminate expression of the gene (ΔflhA), or within the gene following codon 77 (designated flhA77) or codon 454 (designated flhA454), which could allow expression of truncated FlhA proteins. All three flhA mutations severely inhibited transcription of the RpoN-dependent genes flaB and flgE in H. pylori B128. In contrast, levels of flaB and flgE transcripts in H. pylori ATCC 43504 bearing either flhA77 or flhA454, but not ΔflhA, were ~60 % of wild-type levels. The FlhA(454) variant was detected in membrane fractions prepared from H. pylori ATCC 43504 but not H. pylori B128, which may account for the phenotypic differences in the flhA mutations of the two strains. Taken together, these findings suggest that only the N-terminal region of FlhA is needed for transcription of the RpoN regulon. Interestingly, expression of an flaB'-'xylE reporter gene in H. pylori ATCC 43504 bearing the flhA77 allele was about eightfold higher than that of a strain with the wild-type allele, suggesting that expression of flaB is not only regulated at the level of transcription but also regulated post-transcriptionally.

Construction and immunogenicity of a genetically mutant of a native Shigella dysenteriae type 1 isolated from an Iranian patient with diarrhea by recombineering

Since protective immunity against shigellosis is serotype specific, it appears that construction of a live Shigella dysenteriae type 1 strain (attenuated by deletion of the virGgene as a native new vaccine candidate) is urgently needed. The virG gene from a native of S. dysenteriae type 1 was amplified and cloned into the pGEM-5Zf vector and sequenced. Alignment analysis indicated that the virG gene was 100% identical with othershigella serotypes. The polymerase chain reaction fragment carrying a chloramphenicol resistance cassette flanked by short (46 nt) regions homologous to the virG gene was electroporated into recipient S. dysenteriae strains expressing a highly proficient gβexogene system. This cassette allowed successful disruption of one invasive plasmid locus.The results obtained showed that 46-nt homology between the PCR product and the virGgene is enough to promote homologous recombination via the λ Red system. The PCR and sequencing analysis demonstrated that 3220-nt of virG gene was successfully deleted. To intensify recombination efficiency, however, we used 232 ng of purified PCR-product. Upon Sereny test challenge with a native wild S. dysenteriae type 1, ΔvirG-vaccinated animals were significantly protected against keratoconjunctivitis. The λ Red recombinase is a new tool, powerful, inexpensive, rapid and simple method for the construction of in vivo genetic engineering. Key words: Iran, Shigella dysenteriae type 1, virG(icsA), λ red-recombineering,attenuation.

Functional Analysis of ycfR and ycfQ in Escherichia coli O157:H7 Linked to Outbreaks of Illness Associated with Fresh Produce

Fresh produce has been associated with multiple outbreaks of illness caused by Escherichia coli O157:H7. The mechanism of E. coli O157:H7 survival through postharvest processing of fresh produce needs to be understood to help develop more effective interventions. In our recent transcriptomic study of strain Sakai, an isolate from the 1996 sprout outbreak in Japan, and strain TW14359, an isolate from the 2006 spinach outbreak in the United States, we showed that ycfR was the most significantly upregulated gene in response to chlorine-based oxidative stress. YcfR is known to be a multiple stress resistance protein and a biofilm regulator in E. coli K-12 strains; however, its role in the pathogenic E. coli O157:H7 has not been clearly defined. In this study, ycfR was replaced with a chloramphenicol resistance cassette oriented in two different directions to construct polar and nonpolar ycfR::cat mutants of Sakai and TW14359. Chlorine resistance and survival on spinach leaf surfaces were assessed in the wild-type strains and the ycfR mutants. Both polar and nonpolar ycfR mutants of Sakai showed significantly less chlorine resistance than their parent strain. In contrast, deletion of ycfR in TW14359 did not change chlorine resistance, indicating that ycfR in these two outbreak-related E. coli O157:H7 strains may function differently. In addition, after a 24-h incubation on spinach leaves in a sublethal concentration of chlorine, the Sakai nonpolar ycfR mutant exhibited lower survival compared to the wild type. The results suggest a role for ycfR in survival of Sakai during chlorine exposure. We also found that the upstream ycfQ, which is annotated as a DNA-binding regulator, acted as a repressor of ycfR. These findings suggest that gene regulation may be a mechanism by which E. coli O157:H7 strain Sakai could survive in the postharvest processing environment.

Biochemical and Structural Analysis of Bacterial O-antigen Chain Length Regulator Proteins Reveals a Conserved Quaternary Structure

Lipopolysaccharide (LPS) is a major component of the Gram-negative outer membrane and is an important virulence determinant. The O-antigen polysaccharide of the LPS molecule provides protection from host defenses, and the length of O-antigen chains plays a pivotal role. In the Wzy-dependent O-antigen biosynthesis pathway, the integral inner membrane protein Wzz determines the O-antigen chain length. How these proteins function is currently unknown, but the hypothesis includes activities such as a "molecular ruler" or a "molecular stopwatch," and other possibilities may exist. Wzz homologs are membrane proteins with two transmembrane helices that flank a large periplasmic domain. Recent x-ray crystallographic studies of the periplasmic portions of Wzz proteins found multiple oligomeric forms, with quaternary structures favoring the "molecular ruler" interpretation. Here, we have studied full-length Wzz proteins with the transmembrane portions embedded in lipid membranes. Using electron microscopy and image analysis we find a unique hexameric state rather than differing oligomeric forms. The data suggest that in vivo Wzz proteins determine O-antigen chain length via subtle structure-function relationships at the level of primary, secondary, or tertiary structure within the context of a hexameric complex.

The Psb27 Protein Facilitates Manganese Cluster Assembly in Photosystem II

Photosystem II (PSII) is a large membrane protein complex that uses light energy to convert water to molecular oxygen. This enzyme undergoes an intricate assembly process to ensure accurate and efficient positioning of its many components. It has been proposed that the Psb27 protein, a lumenal extrinsic subunit, serves as a PSII assembly factor. Using a psb27 genetic deletion strain (Deltapsb27) of the cyanobacterium Synechocystis sp. PCC 6803, we have defined the role of the Psb27 protein in PSII biogenesis. While the Psb27 protein was not essential for photosynthetic activity, various PSII assembly assays revealed that the Deltapsb27 mutant was defective in integration of the Mn(4)Ca(1)Cl(x) cluster, the catalytic core of the oxygen-evolving machinery within the PSII complex. The other lumenal extrinsic proteins (PsbO, PsbU, PsbV, and PsbQ) are key components of the fully assembled PSII complex and are important for the water oxidation reaction, but we propose that the Psb27 protein has a distinct function separate from these subunits. We show that the Psb27 protein facilitates Mn(4)Ca(1)Cl(x) cluster assembly in PSII at least in part by preventing the premature association of the other extrinsic proteins. Thus, we propose an exchange of lumenal subunits and cofactors during PSII assembly, in that the Psb27 protein is replaced by the other extrinsic proteins upon assembly of the Mn(4)Ca(1)Cl(x) cluster. Furthermore, we show that the Psb27 protein provides a selective advantage for cyanobacterial cells under conditions such as nutrient deprivation where Mn(4)Ca(1)Cl(x) cluster assembly efficiency is critical for survival.

The human gastric pathogen Helicobacter pylori has a potential acetone carboxylase that enhances its ability to colonize mice

BackgroundHelicobacter pylori colonizes the human stomach and is the etiological agent of peptic ulcer disease. All three H. pylori strains that have been sequenced to date contain a potential operon whose products share homology with the subunits of acetone carboxylase (encoded by acxABC) from Xanthobacter autotrophicus strain Py2 and Rhodobacter capsulatus strain B10. Acetone carboxylase catalyzes the conversion of acetone to acetoacetate. Genes upstream of the putative acxABC operon encode enzymes that convert acetoacetate to acetoacetyl-CoA, which is metabolized further to generate two molecules of acetyl-CoA.ResultsTo determine if the H. pylori acxABC operon has a role in host colonization the acxB homolog in the mouse-adapted H. pylori SS1 strain was inactivated with a chloramphenicol-resistance (cat) cassette. In mouse colonization studies the numbers of H. pylori recovered from mice inoculated with the acxB:cat mutant were generally one to two orders of magnitude lower than those recovered from mice inoculated with the parental strain. A statistical analysis of the data using a Wilcoxin Rank test indicated the differences in the numbers of H. pylori isolated from mice inoculated with the two strains were significant at the 99% confidence level. Levels of acetone associated with gastric tissue removed from uninfected mice were measured and found to range from 10–110 μmols per gram wet weight tissue.ConclusionThe colonization defect of the acxB:cat mutant suggests a role for the acxABC operon in survival of the bacterium in the stomach. Products of the H. pylori acxABC operon may function primarily in acetone utilization or may catalyze a related reaction that is important for survival or growth in the host. H. pylori encounters significant levels of acetone in the stomach which it could use as a potential electron donor for microaerobic respiration.

Rapid construction of Campylobacter jejuni deletion mutants

To develop a novel method for rapid construction of Campylobacter jejuni deletion mutants. We used overlapping extension PCR protocol to amplify a target sequence region of Camp. jejuni genomic DNA in which an internal fragment, Cj0618 coding sequence, was replaced by a chloramphenicol resistance cassette. After the resulting PCR product was introduced into electrocompetent Camp. jejuni 81-176, chloramphenicol-resistant mutants in which the wild type allele has been replaced by the deletion cassette were selected. DNA sequencing confirmed precise deletion in the Cj0618 gene. As expected from the previously reported role of Cj0618 in chick colonization, the resulting deletion mutant showed a caecal colonization defect in chick infection. This method can be used for rapid construction of Camp. jejuni deletion mutants. The use of this method should facilitate functional characterization of various Camp. jejuni genes.

Epsilon-Toxin Plasmids of Clostridium perfringens Type D Are Conjugative

Isolates of Clostridium perfringens type D produce the potent epsilon-toxin (a CDC/U.S. Department of Agriculture overlap class B select agent) and are responsible for several economically significant enterotoxemias of domestic livestock. It is well established that the epsilon-toxin structural gene, etx, occurs on large plasmids. We show here that at least two of these plasmids are conjugative. The etx gene on these plasmids was insertionally inactivated using a chloramphenicol resistance cassette to phenotypically tag the plasmid. High-frequency conjugative transfer of the tagged plasmids into the C. perfringens type A strain JIR325 was demonstrated, and the resultant transconjugants were shown to act as donors in subsequent mating experiments. We also demonstrated the transfer of "unmarked" native epsilon-toxin plasmids into strain JIR325 by exploiting the high transfer frequency. The transconjugants isolated in these experiments expressed functional epsilon-toxin since their supernatants had cytopathic effects on MDCK cells and were toxic in mice. Using the widely accepted multiplex PCR approach for toxin genotyping, these type A-derived transconjugants were genotypically type D. These findings have significant implications for the C. perfringens typing system since it is based on the toxin profile of each strain. Our study demonstrated the fluid nature of the toxinotypes and their dependence upon the presence or absence of toxin plasmids, some of which have for the first time been shown to be conjugative.

In Vivo Functional Analyses of the Type II Acyl Carrier Proteins of Fatty Acid Biosynthesis

Acyl carrier protein (ACP) is a key component of the fatty acid synthesis pathways of both type I and type II synthesis systems. A large number of structure-function studies of various type II ACPs have been reported, but all are in vitro studies that assayed function or interaction of mutant ACPs with various enzymes of fatty acid synthesis or transfer. Hence in these studies functional properties of various mutant ACPs were assayed with only a subset of the many ACP-interacting proteins, which may not give an accurate overall view of the function of these proteins in vivo. This is especially so because Escherichia coli ACP has been reported to interact with several proteins that have no known roles in lipid metabolism. We therefore tested a large number of mutant derivatives of E. coli ACP carrying single amino acid substitutions for their abilities to restore growth to an E. coli strain carrying a temperature-sensitive mutation in acpP, the gene that encodes ACP. Many of these mutant proteins had previously been tested in vitro thus providing data for comparison with our results. We found that several mutant ACPs containing substitutions of ACP residues reported previously to be required for ACP function in vitro support normal growth of the acpP mutant strain. However, several mutant proteins reported to be severely defective in vitro failed to support growth of the acpP strain in vivo (or supported only weak growth). A collection of ACPs from diverse bacteria and from three eukaryotic organelles was also tested. All of the bacterial ACPs tested restored growth to the E. coli acpP mutant strain except those from two related bacteria, Enterococcus faecalis and Lactococcus lactis. Only one of the three eukaryotic organellar ACPs allowed growth. Strikingly the ACP is that of the apicoplast of Plasmodium falciparum (the protozoan that causes malaria). The fact that an ACP from a such diverse organism can replace AcpP function in E. coli suggests that some of the protein-protein interactions detected for AcpP may be not be essential for growth of E. coli.