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Identification and characterization of acoK, a regulatory gene of the Klebsiella pneumoniae acoABCD operon.

ABSTRACT: By using transposon insertional mutagenesis and deletion analyses, a recombinant clone containing the region upstream of the acoABCD operon of Klebsiella pneumoniae was found to be required for acetoin-inducible expression of the operon in Escherichia coli. The nucleotide sequence of the region was determined, and it displayed an open reading frame of 2,763 bp that is transcribed divergently to the acoABCD operon. This gene, designated acoK, is capable of encoding a protein with an overall 58.4% amino acid identity with MalT, the transcriptional activator of the E. coli maltose regulon. A conserved sequence for nucleotide binding at the N-terminal region, as well as a helix-turn-helix motif belonging to the LuxR family of transcriptional regulators at the C terminus, was also identified. Primer extension analysis identified two transcription initiation sites, S1 and S2, located 319 and 267 bp, respectively, upstream of the putative start codon of acoK. Several copies of NtrC recognition sequence [CAC-(N11 to N18)-GTG] were found in the promoter regions of both the acoK gene and the acoABCD operon. Acetoin-dependent expression of the acoABCD operon could be restored in the E. coli acoK mutants by supplying a plasmid carrying an intact acoK, suggesting a transactivating function of the gene product. The AcoK protein overproduced in E. coli was approximately 100 kDa, which is in good agreement with the molecular mass deduced from the nucleotide sequence. A specific DNA binding property and an ATPase activity of the purified AcoK were also demonstrated.

SUBMITTER: Peng HL

PROVIDER: S-EPMC178858 | biostudies-other | 1997 Mar

REPOSITORIES: biostudies-other

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Identification and characterization of acoK, a regulatory gene of the Klebsiella pneumoniae acoABCD operon.

Peng H L HL Yang Y H YH Deng W L WL Chang H Y HY

Journal of bacteriology 19970301 5

By using transposon insertional mutagenesis and deletion analyses, a recombinant clone containing the region upstream of the acoABCD operon of Klebsiella pneumoniae was found to be required for acetoin-inducible expression of the operon in Escherichia coli. The nucleotide sequence of the region was determined, and it displayed an open reading frame of 2,763 bp that is transcribed divergently to the acoABCD operon. This gene, designated acoK, is capable of encoding a protein with an overall 58.4% ...[more]

PMID: 9045805

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Project description:BACKGROUND: Several strain-specific Klebsiella pneumoniae virulence determinants have been described, though these have almost exclusively been linked with hypervirulent liver abscess-associated strains. Through PCR interrogation of integration hotspots, chromosome walking, island-tagging and fosmid-based marker rescue we captured and sequenced KpGI-5, a novel genomic island integrated into the met56 tRNA gene of K. pneumoniae KR116, a bloodstream isolate from a patient with pneumonia and neutropenic sepsis. RESULTS: The 14.0 kb KpGI-5 island exhibited a genome-anomalous G +? content, possessed near-perfect 46 bp direct repeats, encoded a ?1-chaperone/usher fimbrial cluster (fim2) and harboured seven other predicted genes of unknown function. Transcriptional analysis demonstrated expression of three fim2 genes, and suggested that the fim2A-fim2K cluster comprised an operon. As fimbrial systems are frequently implicated in pathogenesis, we examined the role of fim2 by analysing KR2107, a streptomycin-resistant derivative of KR116, and three isogenic mutants (?fim, ?fim2 and ?fim?fim2) using biofilm assays, human cell adhesion assays and pair-wise competition-based murine models of intestinal colonization, lung infection and ascending urinary tract infection. Although no statistically significant role for fim2 was demonstrable, liver and kidney CFU counts for lung and urinary tract infection models, respectively, hinted at an ordered gradation of virulence: KR2107 (most virulent), KR2107?fim2, KR2107?fim and KR2107?fim?fim2 (least virulent). Thus, despite lack of statistical evidence there was a suggestion that fim and fim2 contribute additively to virulence in these murine infection models. However, further studies would be necessary to substantiate this hypothesis. CONCLUSION: Although fim2 was present in 13% of Klebsiella spp. strains investigated, no obvious in vitro or in vivo role for the locus was identified, although there were subtle hints of involvement in urovirulence and bacterial dissemination from the respiratory tract. Based on our findings and on parallels with other fimbrial systems, we propose that fim2 has the potential to contribute beneficially to pathogenesis and/or environmental persistence of Klebsiella strains, at least under specific yet-to-be identified conditions.

Project description:Klebsiella pneumoniae is a major cause of nosocomial infection and is considered a clinically important bacterium with antibiotic-resistant strains. There are few reports of K. pneumoniae infections in cultured aquatic animals, and no natural infection has been reported in amphibians. From September to October 2021, a high-mortality disease outbreak occurred in a pond-raised American bullfrog farm in Guangzhou, China. The infected bullfrogs were characterized by multiple organ congestive enlargement and inflammation. A pathogenic bacterium was isolated from the viscera of infected bullfrogs and confirmed to be K. pneumoniae by morphological, biochemical, and phylogenetic analyses. Infection experiments confirmed the virulence of the pathogenic strain against bullfrogs and tadpoles. A histopathological examination showed that the strain was harmful to multiple organs. Antibiotic resistance experiments indicated the isolate was a carbapenemase-producing multidrug-resistant K. pneumoniae (MDR-KP) strain. This study is the first report of K. pneumoniae infected American bullfrogs (Rana catesbeiana) and amphibians. These results will shed light on the pathogenicity of K. pneumoniae and help prevent and control K. pneumoniae infections in bullfrogs. IMPORTANCE Klebsiella pneumoniae is recognized as the most common multidrug-resistant bacterial pathogen in humans, and little is known about its pathogenicity in aquatic animals. Recently, K. pneumoniae was found to cause substantial mortality and morbidity in American farm frogs. This was the first report of K. pneumoniae infecting amphibians. In this study, we analyzed the biochemical, growth, and phylogenetic characteristics of the K. pneumoniae strain and described the symptoms and pathological features of infected bullfrogs and tadpoles; this will provide useful data for the prevention and control of infectious diseases, which has been suggested to decrease economic losses in bullfrog farming and reduce the potential threat to public health posed by K. pneumoniae.

Project description:Klebsiella pneumoniae is found in the environment and as a harmless commensal, but is also a frequent nosocomial pathogen (causing urinary, respiratory and blood infections) and the agent of specific human infections including Friedländer's pneumonia, rhinoscleroma and the emerging disease pyogenic liver abscess (PLA). The identification and precise definition of virulent clones, i.e. groups of strains with a single ancestor that are associated with particular infections, is critical to understand the evolution of pathogenicity from commensalism and for a better control of infections. We analyzed 235 K. pneumoniae isolates of diverse environmental and clinical origins by multilocus sequence typing, virulence gene content, biochemical and capsular profiling and virulence to mice. Phylogenetic analysis of housekeeping genes clearly defined clones that differ sharply by their clinical source and biological features. First, two clones comprising isolates of capsular type K1, clone CC23(K1) and clone CC82(K1), were strongly associated with PLA and respiratory infection, respectively. Second, only one of the two major disclosed K2 clones was highly virulent to mice. Third, strains associated with the human infections ozena and rhinoscleroma each corresponded to one monomorphic clone. Therefore, K. pneumoniae subsp. ozaenae and K. pneumoniae subsp. rhinoscleromatis should be regarded as virulent clones derived from K. pneumoniae. The lack of strict association of virulent capsular types with clones was explained by horizontal transfer of the cps operon, responsible for the synthesis of the capsular polysaccharide. Finally, the reduction of metabolic versatility observed in clones Rhinoscleromatis, Ozaenae and CC82(K1) indicates an evolutionary process of specialization to a pathogenic lifestyle. In contrast, clone CC23(K1) remains metabolically versatile, suggesting recent acquisition of invasive potential. In conclusion, our results reveal the existence of important virulent clones associated with specific infections and provide an evolutionary framework for research into the links between clones, virulence and other genomic features in K. pneumoniae.

Dataset Information

Identification and characterization of acoK, a regulatory gene of the Klebsiella pneumoniae acoABCD operon.

Publications

Identification and characterization of acoK, a regulatory gene of the Klebsiella pneumoniae acoABCD operon.

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