Genetic and transcriptional analysis of a novel plasmid-encoded copper resistance operon from Lactococcus lactis

ABSTRACT To identify components of the copper homeostatic mechanism of Lactococcus lactis, we employed two-dimensional gel electrophoresis to detect changes in the proteome in response to copper. Three proteins upregulated by copper were identified: glyoxylase I (YaiA), a nitroreductase (YtjD), and lactate oxidase (LctO). The promoter regions of these genes feature cop boxes of consensus TACAnnTGTA, which are the binding site of CopY-type copper-responsive repressors. A genome-wide search for cop boxes revealed 28 such sequence motifs. They were tested by electrophoretic mobility shift assays for the interaction with purified CopR, the CopY-type repressor of L. lactis. Seven of the cop boxes interacted with CopR in a copper-sensitive manner. They were present in the promoter region of five genes, lctO, ytjD, copB, ydiD, and yahC; and two polycistronic operons, yahCD-yaiAB and copRZA. Induction of these genes by copper was confirmed by real-time quantitative PCR. The copRZA operon encodes the CopR repressor of the regulon; a copper chaperone, CopZ; and a putative copper ATPase, CopA. When expressed in Escherichia coli, the copRZA operon conferred copper resistance, suggesting that it functions in copper export from the cytoplasm. Other member genes of the CopR regulon may similarly be involved in copper metabolism.

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The Riboflavin Transporter RibU in Lactococcus lactis: Molecular Characterization of Gene Expression and the Transport Mechanism

Journal of Bacteriology ◽

10.1128/jb.188.8.2752-2760.2006 ◽

2006 ◽

Vol 188 (8) ◽

pp. 2752-2760 ◽

Cited By ~ 60

Author(s):

Catherine M. Burgess ◽

Dirk Jan Slotboom ◽

Eric R. Geertsma ◽

Ria H. Duurkens ◽

Bert Poolman ◽

...

Keyword(s):

Lactococcus Lactis ◽

Transcriptional Control ◽

Transport Protein ◽

Transcriptional Analysis ◽

Start Codon ◽

Flavin Mononucleotide ◽

Membrane Spanning ◽

Riboflavin Transport ◽

Classification Database

ABSTRACT This study describes the characterization of the riboflavin transport protein RibU in the lactic acid bacterium Lactococcus lactis subsp. cremoris NZ9000. RibU is predicted to contain five membrane-spanning segments and is a member of a novel transport protein family, not described in the Transport Classification Database. Transcriptional analysis revealed that ribU transcription is downregulated in response to riboflavin and flavin mononucleotide (FMN), presumably by means of the structurally conserved RFN (riboflavin) element located between the transcription start site and the start codon. An L. lactis strain carrying a mutated ribU gene exhibits altered transcriptional control of the riboflavin biosynthesis operon ribGBAH in response to riboflavin and FMN and does not consume riboflavin from its growth medium. Furthermore, it was shown that radiolabeled riboflavin is not taken up by the ribU mutant strain, in contrast to the wild-type strain, directly demonstrating the involvement of RibU in riboflavin uptake. FMN and the toxic riboflavin analogue roseoflavin were shown to inhibit riboflavin uptake and are likely to be RibU substrates. FMN transport by RibU is consistent with the observed transcriptional regulation of the ribGBAH operon by external FMN. The presented transport data are consistent with a uniport mechanism for riboflavin translocation and provide the first detailed molecular and functional analysis of a bacterial protein involved in riboflavin transport.

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Copper Stress Induces a Global Stress Response in Staphylococcus aureus and Represses sae and agr Expression and Biofilm Formation

Applied and Environmental Microbiology ◽

10.1128/aem.02268-09 ◽

2009 ◽

Vol 76 (1) ◽

pp. 150-160 ◽

Cited By ~ 83

Author(s):

Jonathan Baker ◽

Sutthirat Sitthisak ◽

Mrittika Sengupta ◽

Miranda Johnson ◽

R. K. Jayaswal ◽

...

Keyword(s):

Oxidative Stress ◽

Staphylococcus Aureus ◽

Biofilm Formation ◽

Transcriptional Profiling ◽

Resistance Mechanism ◽

Growth Conditions ◽

Copper Resistance ◽

Transcriptional Analysis ◽

Copper Stress ◽

High Copper

ABSTRACT Copper is an important cofactor for many enzymes; however, high levels of copper are toxic. Therefore, bacteria must ensure there is sufficient copper for use as a cofactor but, more importantly, must limit free intracellular levels to prevent toxicity. In this study, we have used DNA microarray to identify Staphylococcus aureus copper-responsive genes. Transcriptional profiling of S. aureus SH1000 grown in excess copper identified a number of genes which fall into four groups, suggesting that S. aureus has four main mechanisms for adapting to high levels of environmental copper, as follows: (i) induction of direct copper homeostasis mechanisms; (ii) increased oxidative stress resistance; (iii) expression of the misfolded protein response; and (iv) repression of a number of transporters and global regulators such as Agr and Sae. Our experimental data confirm that resistance to oxidative stress and particularly to H2O2 scavenging is an important S. aureus copper resistance mechanism. Our previous studies have demonstrated that Eap and Emp proteins, which are positively regulated by Agr and Sae, are required for biofilm formation under low-iron growth conditions. Our transcriptional analysis has confirmed that sae, agr, and eap are repressed under high-copper conditions and that biofilm formation is indeed repressed under high-copper conditions. Therefore, our results may provide an explanation for how copper films can prevent biofilm formation on catheters.

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Copper Resistance of the Emerging Pathogen Acinetobacter baumannii

Applied and Environmental Microbiology ◽

10.1128/aem.01813-16 ◽

2016 ◽

Vol 82 (20) ◽

pp. 6174-6188 ◽

Cited By ~ 26

Author(s):

Caitlin L. Williams ◽

Heather M. Neu ◽

Jeremy J. Gilbreath ◽

Sarah L. J. Michel ◽

Daniel V. Zurawski ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Resistance Genes ◽

Resistance Mechanisms ◽

Copper Resistance ◽

Transcriptional Analysis ◽

Resistant Bacteria ◽

Emerging Pathogen ◽

Amino Acid Homology ◽

Content Type ◽

High Concentrations

ABSTRACTAcinetobacter baumanniiis an important emerging pathogen that is capable of causing many types of severe infection, especially in immunocompromised hosts. SinceA. baumanniican rapidly acquire antibiotic resistance genes, many infections are on the verge of being untreatable, and novel therapies are desperately needed. To investigate the potential utility of copper-based antibacterial strategies againstAcinetobacterinfections, we characterized copper resistance in a panel of recent clinicalA. baumanniiisolates. Exposure to increasing concentrations of copper in liquid culture and on solid surfaces resulted in dose-dependent and strain-dependent effects; levels of copper resistance varied broadly across isolates, possibly resulting from identified genotypic variation among strains. Examination of the growth-phase-dependent effect of copper onA. baumanniirevealed that resistance to copper increased dramatically in stationary phase. Moreover,A. baumanniibiofilms were more resistant to copper than planktonic cells but were still susceptible to copper toxicity. Exposure of bacteria to subinhibitory concentrations of copper allowed them to better adapt to and grow in high concentrations of copper; this copper tolerance response is likely achieved via increased expression of copper resistance mechanisms. Indeed, genomic analysis revealed numerous putative copper resistance proteins that share amino acid homology to known proteins inEscherichia coliandPseudomonas aeruginosa. Transcriptional analysis revealed significant upregulation of these putative copper resistance genes following brief copper exposure. Future characterization of copper resistance mechanisms may aid in the search for novel antibiotics againstAcinetobacterand other highly antibiotic-resistant pathogens.IMPORTANCEAcinetobacter baumanniicauses many types of severe nosocomial infections; unfortunately, some isolates have acquired resistance to almost every available antibiotic, and treatment options are incredibly limited. Copper is an essential nutrient but becomes toxic at high concentrations. The inherent antimicrobial properties of copper give it potential for use in novel therapeutics against drug-resistant pathogens. We show thatA. baumanniiclinical isolates are sensitive to copperin vitro, both in liquid and on solid metal surfaces. Since bacterial resistance to copper is mediated though mechanisms of efflux and detoxification, we identified genes encoding putative copper-related proteins inA. baumanniiand showed that expression of some of these genes is regulated by the copper concentration. We propose that the antimicrobial effects of copper may be beneficial in the development of future therapeutics that target multidrug-resistant bacteria.

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Molecular insights into the copper-sensitive operon repressor in Acidithiobacillus caldus

Applied and Environmental Microbiology ◽

10.1128/aem.00660-21 ◽

2021 ◽

Author(s):

Shaoxiang Hou ◽

Yanjun Tong ◽

Hailin Yang ◽

Shoushuai Feng

Keyword(s):

Binding Affinity ◽

Dissociation Constants ◽

Copper Ions ◽

Copper Resistance ◽

Size Exclusion ◽

Transcriptional Analysis ◽

Copper Stress ◽

Acidithiobacillus Caldus ◽

Competition Assays

The copper-sensitive operon repressor (CsoR) family is the main Cu(I)-sensing family, which is widely distributed, and regulates regulons involved in detoxification in response to extreme copper stress (a general range of ≥ 3 g/L copper ions). Here, we identified CsoR Ac in hyper-copper-resistant Acidithiobacillus caldus , a type strain used in the bioleaching process of copper ores. CsoR Ac possesses highly conserved Cu(I) ligands and structures within the CsoR family members. Transcriptional analysis assays indicated that the promoter (PIII) of csoR was active but weakly responsive to copper in Escherichia coli . Copper titration assays gave a stoichiometry of 0.8 mol Cu(I) per apo-CsoR Ac monomer in vitro combined with atomic absorption spectroscopy analysis. Cu I -CsoR Ac and apo-CsoR Ac share essentially identical secondary structures and assembly states, as demonstrated by circular dichroism spectra and size exclusion chromatography profiles. The average dissociation constants ( K D = 2.26 × 10 −18 M and 0.53 × 10 −15 M) and Cu(I) binding affinity of apo-CsoR Ac were estimated by bathocuproine disulfonate (BCS) and bicinchoninic acid (BCA) competition assays, respectively. Site-directed mutations of conserved Cu(I) ligands in CsoR Ac did not significantly alter the secondary structure or assembly state. Competition assays showed that mutants shared the same order of magnitude of Cu(I) binding affinity with apo-CsoR Ac . Moreover, apo-CsoR Ac could bind to the DNA fragment P08430 in vitro , although with low affinity. Finally, a working model was proposed to illustrate putative copper resistance mechanisms in A. caldus . Importance Research on copper resistance among various species has attracted considerable interest. However, due to the lack of effective and reproducible genetic tools, few studies regarding copper resistance have been reported for A. caldus . Here, we characterized a major Cu(I)-sensing family protein, CsoR Ac , which binds Cu(I) with an attomolar affinity higher than that of the Cu(I)-specific chelator, bathocuproine disulfonate. In particular, CsoR family proteins were only identified in A. caldus , rather than A. ferrooxidans and A. thiooxidans , which are both type strains used for bioleaching. Meanwhile, A. caldus harbored more copper resistance determinants and a relatively full-scale regulatory system involved in copper homeostasis. These observations suggested that A. caldus may play an essential role in the application of engineered strains with higher copper resistance in the near future.

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Metabolic and Transcriptional Analysis of Acid Stress in Lactococcus lactis, with a Focus on the Kinetics of Lactic Acid Pools

PLoS ONE ◽

10.1371/journal.pone.0068470 ◽

2013 ◽

Vol 8 (7) ◽

pp. e68470 ◽

Cited By ~ 23

Author(s):

Ana Lúcia Carvalho ◽

David L. Turner ◽

Luís L. Fonseca ◽

Ana Solopova ◽

Teresa Catarino ◽

...

Keyword(s):

Lactic Acid ◽

Lactococcus Lactis ◽

Acid Stress ◽

Transcriptional Analysis ◽

Kinetics Of

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Sequencing and Transcriptional Analysis of the Biosynthesis Gene Cluster of Putrescine-Producing Lactococcus lactis

Applied and Environmental Microbiology ◽

10.1128/aem.05507-11 ◽

2011 ◽

Vol 77 (18) ◽

pp. 6409-6418 ◽

Cited By ~ 55

Author(s):

Victor Ladero ◽

Fergal P. Rattray ◽

Baltasar Mayo ◽

María Cruz Martín ◽

María Fernández ◽

...

Keyword(s):

Lactococcus Lactis ◽

Regulatory Gene ◽

Transcriptional Analysis ◽

Biosynthesis Gene Cluster ◽

Content Type ◽

Catabolic Enzymes ◽

History Of ◽

History Of Use ◽

Flanking Regions

ABSTRACTLactococcus lactisis a prokaryotic microorganism with great importance as a culture starter and has become the model species among the lactic acid bacteria. The long and safe history of use ofL. lactisin dairy fermentations has resulted in the classification of this species as GRAS (General Regarded As Safe) or QPS (Qualified Presumption of Safety). However, our group has identified several strains ofL. lactissubsp.lactisandL. lactissubsp.cremoristhat are able to produce putrescine from agmatine via the agmatine deiminase (AGDI) pathway. Putrescine is a biogenic amine that confers undesirable flavor characteristics and may even have toxic effects. The AGDI cluster ofL. lactisis composed of a putative regulatory gene,aguR, followed by the genes (aguB,aguD,aguA, andaguC) encoding the catabolic enzymes. These genes are transcribed as an operon that is induced in the presence of agmatine. In some strains, an insertion (IS) element interrupts the transcription of the cluster, which results in a non-putrescine-producing phenotype. Based on this knowledge, a PCR-based test was developed in order to differentiate nonproducingL. lactisstrains from those with a functional AGDI cluster. The analysis of the AGDI cluster and their flanking regions revealed that the capacity to produce putrescine via the AGDI pathway could be a specific characteristic that was lost during the adaptation to the milk environment by a process of reductive genome evolution.

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Lactococcus lactis fabH, Encoding β-Ketoacyl-Acyl Carrier Protein Synthase, Can Be Functionally Replaced by the Plasmodium falciparum Congener

Applied and Environmental Microbiology ◽

10.1128/aem.00170-10 ◽

2010 ◽

Vol 76 (12) ◽

pp. 3959-3966 ◽

Cited By ~ 3

Author(s):

Yu Du ◽

Jolyn E. Gisselberg ◽

Jacob D. Johnson ◽

Patricia J. Lee ◽

Sean T. Prigge ◽

...

Keyword(s):

Fatty Acids ◽

Plasmodium Falciparum ◽

Fatty Acid ◽

Lactococcus Lactis ◽

Fatty Acid Synthase ◽

Acyl Carrier Protein ◽

Transcriptional Analysis ◽

Carrier Protein ◽

Cell Extracts ◽

Fas Ii

ABSTRACT Plasmodium falciparum, in addition to scavenging essential fatty acids from its intra- and intercellular environments, possesses a functional complement of type II fatty acid synthase (FAS) enzymes targeted to the apicoplast organelle. Recent evidence suggests that products of the plasmodial FAS II system may be critical for the parasite's liver-to-blood cycle transition, and it has been speculated that endogenously generated fatty acids may be precursors for essential cofactors, such as lipoate, in the apicoplast. β-Ketoacyl-acyl carrier protein (ACP) synthase III (pfKASIII or FabH) is one of the key enzymes in the initiating steps of the FAS II pathway, possessing two functions in P. falciparum: the decarboxylative thio-Claisen condensation of malonyl-ACP and various acyl coenzymes A (acyl-CoAs; KAS activity) and the acetyl-CoA:ACP transacylase reaction (ACAT). Here, we report the generation and characterization of a hybrid Lactococcus lactis strain that translates pfKASIII instead of L. lactis f abH to initiate fatty acid biosynthesis. The L. lactis expression vector pMG36e was modified for the efficient overexpression of the plasmodial gene in L. lactis. Transcriptional analysis indicated high-efficiency overexpression, and biochemical KAS and ACAT assays confirm these activities in cell extracts. Phenotypically, the L. lactis strain expressing pfKASIII has a growth rate and fatty acid profiles that are comparable to those of the strain complemented with its endogenous gene, suggesting that pfKASIII can use L. lactis ACP as substrate and perform near-normal function in L. lactis cells. This strain may have potential application as a bacterial model for pfKASIII inhibitor prescreening.

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