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Expression of Escherichia coli araE and modified lacY genes in Campylobacter jejuni is not sufficient for arabinose transport.

ABSTRACT: Introduction:Unlike Escherichia coli , Campylobacter jejuni is unable to import a range of sugars, including arabinose, which makes common expression vectors, such as pBAD33, non-functional in these bacteria. Aim:The aim of this study was to investigate whether the E. coli transporters AraE and modified LacY (LacYA177C) would enable C. jejuni to uptake arabinose. Methodology and Results:The respective genes of E. coli were constitutively expressed in C. jejuni strain 11168H after integration into the chromosome via homologous recombination. Vectors carrying these genes also contained a reporter gene, gfp, under the control of the arabinose-inducible promoter, pBAD. These constructs were verified in E. coli by demonstrating the induction of gfp in the presence of arabinose. Integration of the genes into one of the rRNA gene clusters was verified by PCR and genome sequencing. The latter also confirmed that the inserted gene clusters contained no mutations. Expression of the gfp gene in the presence of arabinose inducer was monitored using fluorescence microscopy of colonies and fluorimetry using both whole cells and lysates. Conclusion:The results demonstrated the inability of C. jejuni to use arabinose transporters, which are fully functional in E. coli , suggesting a remarkable difference in the physiology of these bacteria.

SUBMITTER: Ramesh A

PROVIDER: S-EPMC7470287 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Expression of <i>Escherichia coli araE</i> and modified <i>lacY</i> genes in <i>Campylobacter jejuni</i> is not sufficient for arabinose transport.

Ramesh Amritha A Ikeda Naomi N Rubinchik Sona S Karlyshev Andrey V AV

Access microbiology 20190716 5

<h4>Introduction</h4>Unlike <i>Escherichia coli</i> , <i>Campylobacter jejuni</i> is unable to import a range of sugars, including arabinose, which makes common expression vectors, such as pBAD33, non-functional in these bacteria.<h4>Aim</h4>The aim of this study was to investigate whether the <i>E. coli</i> transporters AraE and modified LacY (LacYA177C) would enable <i>C. jejuni</i> to uptake arabinose.<h4>Methodology and results</h4>The respective genes of <i>E. coli</i> were constitutively e ...[more]

PMID: 32974528

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Project description:BACKGROUND:Campylobacter jejuni and Campylobacter coli are major global causes of bacterial gastroenteritis. Whilst several individual colonisation and virulence factors have been identified, our understanding of their role in the transmission, pathogenesis and ecology of Campylobacter has been hampered by the genotypic and phenotypic diversity within C. jejuni and C. coli. Autotransporter proteins are a family of outer membrane or secreted proteins in Gram-negative bacteria such as Campylobacter, which are associated with virulence functions. In this study we have examined the distribution and predicted functionality of the previously described capC and the newly identified, related capD autotransporter gene families in Campylobacter. RESULTS:Two capC-like autotransporter families, designated capC and capD, were identified by homology searches of genomes of the genus Campylobacter. Each family contained four distinct orthologs of CapC and CapD. The distribution of these autotransporter genes was determined in 5829 C. jejuni and 1347 C. coli genomes. Autotransporter genes were found as intact, complete copies and inactive formats due to premature stop codons and frameshift mutations. Presence of inactive and intact autotransporter genes was associated with C. jejuni and C. coli multi-locus sequence types, but for capC, inactivation was independent from the length of homopolymeric tracts in the region upstream of the capC gene. Inactivation of capC or capD genes appears to represent lineage-specific gene decay of autotransporter genes. Intact capC genes were predominantly associated with the C. jejuni ST-45 and C. coli ST-828 generalist lineages. The capD3 gene was only found in the environmental C. coli Clade 3 lineage. These combined data support a scenario of inter-lineage and interspecies exchange of capC and subsets of capD autotransporters. CONCLUSIONS:In this study we have identified two novel, related autotransporter gene families in the genus Campylobacter, which are not uniformly present and exhibit lineage-specific associations and gene decay. The distribution and decay of the capC and capD genes exemplifies the erosion of species barriers between certain lineages of C. jejuni and C. coli, probably arising through co-habitation. This may have implications for the phenotypic variability of these two pathogens and provide opportunity for new, hybrid genotypes to emerge.

Project description:BACKGROUND:Campylobacter is one of the leading bacterial species causing foodborne illnesses in humans. Antimicrobial agents have been extensively used for treatment of Campylobacter infections; but in the recent years, both animal and human isolates of this bacterium have shown resistance to several antibiotics such as tetracycline. OBJECTIVES:The aim of this study was to investigate the presence of genetic determinants of tetracycline resistance in Campylobacter spp. recovered from poultry carcasses in Shiraz, Iran. MATERIALS AND METHODS:Eighty-three thermophilic Campylobacter spp. Isolates were first identified based on multiplex polymerase chain reaction (PCR) and then screened for presence of tetracycline resistance genes (tet (A), tet (B), tet (O) and te (S)) by PCR. RESULTS:The overall prevalence of Campylobacter jejuni and C. coli among the examined isolates was 51.8% and 48.2%, respectively. Tetracycline resistance genes of tet (B) and tet (S) were not seen among these Campylobacter spp. Isolates, whereas the most common tet gene identi?ed was tet (O), found in 83.1% (69/83) of all the isolates. The tet (O) gene sequence comparison between C. jejuni and C. coli showed 100% similarity and these sequences (JX853721and JX853722) were also identical to the homologous sequences of other strains of Campylobacter spp. existing in the GenBank databases. In addition, tet (A) was found in 18% (15/83) of Campylobacter spp. isolates. To our knowledge, this represents the first report of tet (A) in Campylobacter spp. There was 100% homology between the sequences of tet (A) from this study (JX891463 and JX891464) and the tet (A) sequences mentioned for other bacteria in the GenBank databases. CONCLUSIONS:The high prevalence of tet (O) resistance gene along with new detection of tet (A) resistance gene in Campylobacter spp. isolated from poultry carcasses revealed an extensive tetracycline resistance among Campylobacter isolates from poultry in Iran. It emphasized the need for cautious use of tetracycline in poultry production to decrease the extension of tetracycline-resistant Campylobacter spp.

Dataset Information

Expression of Escherichia coli araE and modified lacY genes in Campylobacter jejuni is not sufficient for arabinose transport.

Publications

Expression of <i>Escherichia coli araE</i> and modified <i>lacY</i> genes in <i>Campylobacter jejuni</i> is not sufficient for arabinose transport.

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