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1.
Microbiol Spectr ; : e0062424, 2024 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-38958463

RESUMO

Growing numbers of infections caused by antibiotic-resistant Streptococcus pneumoniae strains are a major concern for healthcare systems that will require new antibiotics for treatment as well as preventative measures that reduce the number of infections. Lipopeptides are antimicrobial molecules, of which some are used as antibiotics, including the last resort antibiotics daptomycin and polymyxins. Here we have studied the antimicrobial effect of the cyclic lipopeptide viscosin on S. pneumoniae growth and morphology. Most lipopeptides function as surfactants that create pores in membrane layers, which is regarded as their main antimicrobial activity. We show that viscosin can inhibit growth of S. pneumoniae without disintegration of the cytoplasmic membrane. Instead, the cells developed abnormal shapes and misplaced new division sites. The cell wall of these bacteria appeared less dense in electron microscopy images, suggesting that viscosin interfered with normal cell wall synthesis. Corroborating this observation, a luciferase reporter assay was used to show that the two-component systems LiaFSR and CiaRH, which are known to be activated upon cell wall stress, were strongly induced by viscosin. Furthermore, a mutant displaying 1.8-fold decreased susceptibility to viscosin was generated by sequential exposure to increasing concentrations of the lipopeptide. The mutant suffered from significant fitness loss and had mutations in genes involved in fatty acid synthesis, teichoic acid synthesis, and cell wall synthesis as well as transcription and translation. How these mutations might be linked to decreased viscosin susceptibility is discussed.IMPORTANCEStreptococcus pneumoniae is a leading cause of bacterial pneumonia, sepsis, and meningitis in children, and the incidence of infections caused by antibiotic-resistant strains is increasing. Development of new antibiotics is therefore necessary to treat these types of infections in the future. Here, we have studied the activity of the antimicrobial lipopeptide viscosin on S. pneumoniae and show that in addition to having the typical membrane destabilizing activity of lipopeptides, viscosin inhibits pneumococcal growth by obstructing normal cell wall synthesis. This suggests a more specific mode of action than just the surfactant activity. Furthermore, we show that S. pneumoniae does not easily acquire resistance to viscosin, which makes it a promising molecule to explore further, for example, by synthesizing less toxic derivates that can be tested for therapeutic potential.

2.
mSystems ; : e0128923, 2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38837392

RESUMO

Antibiotic resistance and tolerance remain a major problem for the treatment of staphylococcal infections. Identifying genes that influence antibiotic susceptibility could open the door to novel antimicrobial strategies, including targets for new synergistic drug combinations. Here, we developed a genome-wide CRISPR interference library for Staphylococcus aureus, demonstrated its use by quantifying gene fitness in different strains through CRISPRi-seq, and used it to identify genes that modulate susceptibility to the lipoglycopeptide dalbavancin. By exposing the library to sublethal concentrations of dalbavancin using both CRISPRi-seq and direct selection methods, we not only found genes previously reported to be involved in antibiotic susceptibility but also identified genes thus far unknown to affect antibiotic tolerance. Importantly, some of these genes could not have been detected by more conventional transposon-based knockout approaches because they are essential for growth, stressing the complementary value of CRISPRi-based methods. Notably, knockdown of a gene encoding the uncharacterized protein KapB specifically sensitizes the cells to dalbavancin, but not to other antibiotics of the same class, whereas knockdown of the Shikimate pathway showed the opposite effect. The results presented here demonstrate the promise of CRISPRi-seq screens to identify genes and pathways involved in antibiotic susceptibility and pave the way to explore alternative antimicrobial treatments through these insights.IMPORTANCEAntibiotic resistance is a challenge for treating staphylococcal infections. Identifying genes that affect how antibiotics work could help create new treatments. In our study, we made a CRISPR interference library for Staphylococcus aureus and used this to find which genes are critical for growth and also mapped genes that are important for antibiotic sensitivity, focusing on the lipoglycopeptide antibiotic dalbavancin. With this method, we identified genes that altered the sensitivity to dalbavancin upon knockdown, including genes involved in different cellular functions. CRISPRi-seq offers a means to uncover untapped antibiotic targets, including those that conventional screens would disregard due to their essentiality. This paves the way for the discovery of new ways to fight infections.

3.
mBio ; 15(6): e0115724, 2024 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-38757970

RESUMO

Coordinated membrane and cell wall synthesis is vital for maintaining cell integrity and facilitating cell division in bacteria. However, the molecular mechanisms that underpin such coordination are poorly understood. Here we uncover the pivotal roles of the staphylococcal proteins CozEa and CozEb, members of a conserved family of membrane proteins previously implicated in bacterial cell division, in the biosynthesis of lipoteichoic acids (LTA) and maintenance of membrane homeostasis in Staphylococcus aureus. We establish that there is a synthetic lethal relationship between CozE and UgtP, the enzyme synthesizing the LTA glycolipid anchor Glc2DAG. By contrast, in cells lacking LtaA, the flippase of Glc2DAG, the essentiality of CozE proteins was alleviated, suggesting that the function of CozE proteins is linked to the synthesis and flipping of the glycolipid anchor. CozE proteins were indeed found to modulate the flipping activity of LtaA in vitro. Furthermore, CozEb was shown to control LTA polymer length and stability. Together, these findings establish CozE proteins as novel players in membrane homeostasis and LTA biosynthesis in S. aureus.IMPORTANCELipoteichoic acids are major constituents of the cell wall of Gram-positive bacteria. These anionic polymers are important virulence factors and modulators of antibiotic susceptibility in the important pathogen Staphylococcus aureus. They are also critical for maintaining cell integrity and facilitating proper cell division. In this work, we discover that a family of membrane proteins named CozE is involved in the biosynthesis of lipoteichoic acids (LTAs) in S. aureus. CozE proteins have previously been shown to affect bacterial cell division, but we here show that these proteins affect LTA length and stability, as well as the flipping of glycolipids between membrane leaflets. This new mechanism of LTA control may thus have implications for the virulence and antibiotic susceptibility of S. aureus.


Assuntos
Proteínas de Bactérias , Lipopolissacarídeos , Proteínas de Membrana , Staphylococcus aureus , Ácidos Teicoicos , Ácidos Teicoicos/biossíntese , Ácidos Teicoicos/metabolismo , Staphylococcus aureus/metabolismo , Staphylococcus aureus/genética , Lipopolissacarídeos/biossíntese , Lipopolissacarídeos/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Parede Celular/metabolismo , Membrana Celular/metabolismo
4.
Microbiology (Reading) ; 169(3)2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36881456

RESUMO

Infections caused by antibiotic-resistant Streptococcus pneumoniae are of growing concern for healthcare systems, which need new treatment options. Screening microorganisms in terrestrial environments has proved successful for discovering antibiotics, while production of antimicrobials by marine microorganisms remains underexplored. Here we have screened microorganisms sampled from the Oslo Fjord in Norway for production of molecules that prevent the human pathogen S. pneumoniae from growing. A bacterium belonging to the genus Lysinibacillus was identified. We show that this bacterium produces a molecule that kills a wide range of streptococcal species. Genome mining in BAGEL4 and AntiSmash suggested that it was a new antimicrobial compound, and we therefore named it lysinicin OF. The compound was resistant to heat (100 °C) and polymyxin acylase but susceptible to proteinase K, showing that it is of proteinaceous nature, but most probably not a lipopeptide. S. pneumoniae became resistant to lysinicin OF by obtaining suppressor mutations in the ami locus, which encodes the AmiACDEF oligo peptide transporter. We created ΔamiC and ΔamiEF mutants to show that pneumococci expressing a compromised Ami system were resistant to lysinicin OF. Furthermore, by creating mutants expressing an intact but inactive Ami system (AmiED184A and AmiFD175A) we could conclude that the lysinicin OF activity depended on the active form (ATP-hydrolysing) of the Ami system. Microscopic imaging and fluorescent labelling of DNA showed that S. pneumoniae treated with lysinicin OF had an average reduced cell size with condensed DNA nucleoid, while the integrity of the cell membrane remained intact. The characteristics and possible mode of action of lysinicin OF are discussed.


Assuntos
Bacillaceae , Streptococcus pneumoniae , Humanos , Streptococcus pneumoniae/genética , Bacillaceae/genética , Oligopeptídeos , Antibacterianos/farmacologia , Membrana Celular
5.
mBio ; 13(2): e0340421, 2022 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-35357211

RESUMO

Cell division and cell wall synthesis in staphylococci need to be precisely coordinated and controlled to allow the cell to multiply while maintaining its nearly spherical shape. The mechanisms ensuring correct placement of the division plane and synthesis of new cell wall have been studied intensively. However, hitherto unknown factors and proteins are likely to play key roles in this complex interplay. Here, we identified and investigated a protein with a major influence on cell morphology in Staphylococcus aureus. The protein, named SmdA (for staphylococcal morphology determinant A), is a membrane protein with septum-enriched localization. By CRISPRi knockdown and overexpression combined with different microscopy techniques, we demonstrated that proper levels of SmdA were necessary for cell division, including septum formation and cell splitting. We also identified conserved residues in SmdA that were critical for its functionality. Pulldown and bacterial two-hybrid interaction experiments showed that SmdA interacted with several known cell division and cell wall synthesis proteins, including penicillin-binding proteins (PBPs) and EzrA. Notably, SmdA also affected susceptibility to cell wall targeting antibiotics, particularly in methicillin-resistant S. aureus (MRSA). Together, our results showed that S. aureus was dependent on balanced amounts of membrane attached SmdA to carry out proper cell division. IMPORTANCE Staphylococcus aureus is an important human and animal pathogen. Antibiotic resistance is a major problem in the treatment of staphylococcal infections, and cell division and cell wall synthesis factors have previously been shown to modulate susceptibility to antibiotics in this species. Here, we investigated the function of a protein named SmdA, which was identified based on its septal localization and knockdown phenotype resulting in defective cellular morphologies. We demonstrated that this protein was critical for normal cell division in S. aureus. Depletion of SmdA sensitized resistant staphylococci to ß-lactam antibiotics. This work revealed a new staphylococcal cell division factor and a potential future target for narrow-spectrum antimicrobials or compounds to resensitize antibiotic-resistant staphylococcal strains.


Assuntos
Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Humanos , Staphylococcus aureus Resistente à Meticilina/metabolismo , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/metabolismo , Staphylococcus/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo
6.
Microbiologyopen ; 9(8): e1057, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32419377

RESUMO

Methicillin-resistant Staphylococcus aureus (MRSA) is resistant to most ß-lactams due to the expression of an extra penicillin-binding protein, PBP2a, with low ß-lactam affinity. It has long been known that heterologous expression of the PBP2a-encoding mecA gene in methicillin-sensitive S. aureus (MSSA) provides protection towards ß-lactams, however, some reports suggest that the degree of protection can vary between different ß-lactams. To test this more systematically, we introduced an IPTG-inducible mecA into the MSSA laboratory strain RN4220. We confirm, by growth assays as well as single-cell microfluidics time-lapse microscopy experiments, that PBP2a expression protects against ß-lactams in S. aureus RN4220. By testing a panel of ten different ß-lactams, we conclude that there is also a great variation in the level of protection conferred by PBP2a. Expression of PBP2a resulted in an only fourfold increase in minimum inhibitory concentration (MIC) for imipenem, while a 32-fold increase in MIC was observed for cefaclor and cephalexin. Interestingly, in our experimental setup, PBP2a confers the highest protection against cefaclor and cephalexin-two ß-lactams that are known to have a high specific affinity toward the transpeptidase PBP3 of S. aureus. Notably, using a single-cell microfluidics setup we demonstrate a considerable phenotypic variation between cells upon ß-lactam exposure and show that mecA-expressing S. aureus can survive ß-lactam concentrations much higher than the minimal inhibitory concentrations. We discuss possible explanations and implications of these results including important aspects regarding treatment of infection.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/genética , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Proteínas de Ligação às Penicilinas/genética , beta-Lactamas/farmacologia , Proteínas de Bactérias/metabolismo , Humanos , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Testes de Sensibilidade Microbiana , Microfluídica , Proteínas de Ligação às Penicilinas/metabolismo
7.
mSphere ; 4(2)2019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30894429

RESUMO

Studies of essential genes in bacteria are often hampered by the lack of accessible genetic tools. This is also the case for Lactobacillus plantarum, a key species in food and health applications. Here, we develop a clustered regularly interspaced short palindromic repeat interference (CRISPRi) system for knockdown of gene expression in L. plantarum The two-plasmid CRISPRi system, in which a nuclease-inactivated Cas9 (dCas9) and a gene-specific single guide RNA (sgRNA) are expressed on separate plasmids, allows efficient knockdown of expression of any gene of interest. We utilized the CRISPRi system to gain initial insights into the functions of key cell cycle genes in L. plantarum As a proof of concept, we investigated the phenotypes resulting from knockdowns of the cell wall hydrolase-encoding acm2 gene and of the DNA replication initiator gene dnaA and of ezrA, which encodes an early cell division protein. Furthermore, we studied the phenotypes of three cell division genes which have recently been functionally characterized in ovococcal bacteria but whose functions have not yet been investigated in rod-shaped bacteria. We show that the transmembrane CozE proteins do not seem to play any major role in cell division in L. plantarum On the other hand, RNA-binding proteins KhpA and EloR are critical for proper cell elongation in this bacterium.IMPORTANCEL. plantarum is an important bacterium for applications in food and health. Deep insights into the biology and physiology of this species are therefore necessary for further strain optimization and exploitation; however, the functions of essential genes in the bacterium are mainly unknown due to the lack of accessible genetic tools. The CRISPRi system developed here is ideal to quickly screen for phenotypes of both essential and nonessential genes. Our initial insights into the function of some key cell cycle genes represent the first step toward understanding the cell cycle in this bacterium.


Assuntos
Sistemas CRISPR-Cas , Técnicas de Silenciamento de Genes/métodos , Genes Essenciais , Genes cdc , Lactobacillus plantarum/genética , Proteínas de Bactérias/genética , Parede Celular/enzimologia , Parede Celular/genética , Replicação do DNA , Fenótipo , Plasmídeos/genética , Interferência de RNA
8.
Mol Microbiol ; 109(5): 615-632, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29884993

RESUMO

Staphylococcus aureus needs to control the position and timing of cell division and cell wall synthesis to maintain its spherical shape. We identified two membrane proteins, named CozEa and CozEb, which together are important for proper cell division in S. aureus. CozEa and CozEb are homologs of the cell elongation regulator CozESpn of Streptococcus pneumoniae. While cozEa and cozEb were not essential individually, the ΔcozEaΔcozEb double mutant was lethal. To study the functions of cozEa and cozEb, we constructed a CRISPR interference (CRISPRi) system for S. aureus, allowing transcriptional knockdown of essential genes. CRISPRi knockdown of cozEa in the ΔcozEb strain (and vice versa) causes cell morphological defects and aberrant nucleoid staining, showing that cozEa and cozEb have overlapping functions and are important for normal cell division. We found that CozEa and CozEb interact with and possibly influence localization of the cell division protein EzrA. Furthermore, the CozE-EzrA interaction is conserved in S. pneumoniae, and cell division is mislocalized in cozESpn -depleted S. pneumoniae cells. Together, our results show that CozE proteins mediate control of cell division in S. aureus and S. pneumoniae, likely via interactions with key cell division proteins such as EzrA.


Assuntos
Proteínas de Bactérias/fisiologia , Proteínas de Ciclo Celular/metabolismo , Divisão Celular/genética , Proteínas de Membrana/fisiologia , Staphylococcus aureus/fisiologia , Proteínas de Bactérias/genética , Proteínas de Ciclo Celular/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA Bacteriano/genética , Técnicas de Silenciamento de Genes , Proteínas de Membrana/genética , Mutação , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade
9.
Mol Microbiol ; 103(1): 99-116, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27684385

RESUMO

The oval shape of pneumococci results from a combination of septal and lateral peptidoglycan synthesis. The septal cross-wall is synthesized by the divisome, while the elongasome drives cell elongation by inserting new peptidoglycan into the lateral cell wall. Each of these molecular machines contains penicillin-binding proteins (PBPs), which catalyze the final stages of peptidoglycan synthesis, plus a number of accessory proteins. Much effort has been made to identify these accessory proteins and determine their function. In the present paper we have used a novel approach to identify members of the pneumococcal elongasome that are functionally closely linked to PBP2b. We discovered that cells depleted in PBP2b, a key component of the elongasome, display several distinct phenotypic traits. We searched for proteins that, when depleted or deleted, display the same phenotypic changes. Four proteins, RodA, MreD, DivIVA and Spr0777, were identified by this approach. Together with PBP2b these proteins are essential for the normal function of the elongasome. Furthermore, our findings suggest that DivIVA, which was previously assigned as a divisomal protein, is required to correctly localize the elongasome at the negatively curved membrane region between the septal and lateral cell wall.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/metabolismo , Sequência de Aminoácidos , Aminoaciltransferases/metabolismo , Proteínas de Transporte/metabolismo , Crescimento Celular , Parede Celular/metabolismo , Proteínas de Membrana/metabolismo , Testes de Sensibilidade Microbiana , Penicilinas , Peptidoglicano/biossíntese , Peptidil Transferases/metabolismo , Streptococcus pneumoniae/metabolismo , Técnicas do Sistema de Duplo-Híbrido
10.
Microbiology (Reading) ; 163(1): 9-21, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27902435

RESUMO

The important human pathogen Streptococcus pneumoniae is a naturally transformable species. When developing the competent state, it expresses proteins involved in DNA uptake, DNA processing and homologous recombination. In addition to the proteins required for the transformation process, competent pneumococci express proteins involved in a predatory DNA acquisition mechanism termed fratricide. This is a mechanism by which the competent pneumococci secrete a muralytic fratricin termed CbpD, which lyses susceptible sister cells or closely related streptococcal species. The released DNA can then be taken up by the competent pneumococci and integrated into their genomes. To avoid committing suicide, competent pneumococci produce an integral membrane protein, ComM, which protects them against CbpD by an unknown mechanism. In the present study, we show that overexpression of ComM results in growth inhibition and development of severe morphological abnormalities, such as cell elongation, misplacement of the septum and inhibition of septal cross-wall synthesis. The toxic effect of ComM is tolerated during competence because it is not allowed to accumulate in the competent cells. We provide evidence that an intra-membrane protease called RseP is involved in the process of controlling the ComM levels, since △rseP mutants produce higher amounts of ComM compared to wild-type cells. The data presented here indicate that ComM mediates immunity against CbpD by a mechanism that is detrimental to the pneumococcus if exaggerated.


Assuntos
Amidoidrolases/metabolismo , Proteínas de Bactérias/biossíntese , Bacteriólise/fisiologia , Competência de Transformação por DNA/genética , Proteínas de Membrana/biossíntese , Peptídeo Hidrolases/metabolismo , Streptococcus pneumoniae/crescimento & desenvolvimento , Amidoidrolases/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , DNA Bacteriano/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Peptídeo Hidrolases/genética , Peptidoglicano/biossíntese , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/patogenicidade , Transformação Bacteriana/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
11.
Microbiology (Reading) ; 163(3): 383-399, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27902439

RESUMO

WalRK is the only two-component regulatory system essential for viability in Streptococcus pneumoniae. Despite its importance, the biological role of this system is not well understood. However, previous studies have shown that it has a crucial role in controlling pneumococcal cell division. Considerable efforts have been made to understand how the WalRK system is regulated, but no signal(s) sensed by the WalK histidine kinase has been identified so far. Here, we provide evidence that the serine/threonine protein kinase StkP modulates the activity of WalK through direct protein-protein interaction, suggesting that this interaction is one of the signals sensed by WalK. In most low-G+C content Gram-positive bacteria, WalK orthologues are attached to the cytoplasmic membrane via two transmembrane segments separated by a large extracellular loop believed to function as a sensor domain. In contrast, members of the genus Streptococcus have WalK histidine kinases that are anchored to the cytoplasmic membrane by a single transmembrane segment. It has been a long-standing question whether this segment only serves as a membrane anchor or if it also functions as a signal-sensing domain. Our data strongly support the latter, i.e. that the transmembrane segment senses signals that regulate the activity of WalK.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Streptococcus pneumoniae/metabolismo , Sequência de Aminoácidos/genética , Proteínas de Bactérias/genética , Composição de Bases/genética , Divisão Celular/genética , Membrana Celular/metabolismo , Domínios Proteicos/genética , Transdução de Sinais
12.
J Bacteriol ; 196(5): 911-9, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24336941

RESUMO

Garvicin ML (GarML) is a circular bacteriocin produced by Lactococcus garvieae DCC43. The recently published draft genome of this strain allowed determination of the genetic background for bacteriocin production. Bioinformatic analysis identified a gene cluster consisting of nine open reading frames likely involved in the production of and immunity to GarML. The garA gene encodes the bacteriocin precursor, garX a large transmembrane protein, garBCDE a putative immunity protein (garB) followed by an ATPase and two transmembrane proteins, and garFGH a putative ABC transporter complex. Functional genetic analysis revealed that deletion of garFGH had no effect on sensitivity to or production of GarML. In contrast, deletion of garBCDE or inactivation of garX resulted in high-level sensitivity to GarML and completely abolished production of active bacteriocin. Mass spectrometry of culture supernatants revealed that wild-type cultures contained the mature circular form as well as the linear forms of the bacteriocin, both with and without the three-amino-acid leader sequence, while bacteriocin-negative mutants contained only the linear forms. These results indicate that cleavage of the leader peptide precedes circularization and is likely performed by a functional entity separate from the GarML gene cluster. To our knowledge, this is the first conclusive evidence for these processes being separated in time. Loss of immunity and antimicrobial activity in addition to our inability to detect the circular bacteriocin in the ΔgarBCDE and garX::pCG47 mutants demonstrate that both these units are indispensable for GarML biosynthesis as well as immunity. Furthermore, the results indicate that these genes are implicated in the circularization of the bacteriocin and that their functions are probably interlinked.


Assuntos
Bacteriocinas/biossíntese , Regulação Bacteriana da Expressão Gênica/fisiologia , Lactococcus/classificação , Lactococcus/genética , Bacteriocinas/genética , Bacteriocinas/metabolismo , Clonagem Molecular , Biologia Computacional , Genótipo , Dados de Sequência Molecular , Família Multigênica
13.
J Bacteriol ; 194(4): 875-83, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22155775

RESUMO

Aureocin A53 is an antimicrobial peptide produced by Staphylococcus aureus A53. The genetic determinants involved in aureocin A53 production and immunity to its action are organized in at least four transcriptional units encoded by the 10.4-kb plasmid pRJ9. One transcriptional unit carries only the bacteriocin structural gene, aucA. No immunity gene is found downstream of aucA, as part of the same transcriptional unit. Further downstream of aucA is found an operon which contains the three genes aucEFG, whose products seem to associate to form a dedicated ABC transporter. When aucEFG were expressed in RN4220, an aureocin A53-sensitive S. aureus strain, this strain became partially resistant to the bacteriocin. A gene disruption mutant in aucE was defective in aureocin A53 externalization and more sensitive to aureocin A53 than the wild-type strain, showing that aucEFG are involved in immunity to aureocin A53 by active extrusion of the bacteriocin. Full resistance to aureocin A53 was exhibited by transformants carrying, besides aucEFG, the operon formed by two genes, aucIB and aucIA, located between aucA and aucEFG and carried in the opposite strand. AucIA and AucIB share similarities with hypothetical proteins not found in the gene clusters of other bacteriocins. A gene disruption mutant in orf8, located upstream of aucA and whose product exhibits about 50% similarity to a number of hypothetical membrane proteins found in many Gram-positive bacteria, was strongly affected in aureocin A53 externalization but resistant to aureocin A53, suggesting that Orf8 is also involved in aureocin A53 secretion.


Assuntos
Bacteriocinas/genética , Bacteriocinas/metabolismo , Genes Bacterianos , Peptídeos/genética , Peptídeos/metabolismo , Staphylococcus aureus/genética , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Peptídeos Catiônicos Antimicrobianos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriocinas/química , Bacteriocinas/farmacologia , Sequência de Bases , Escherichia coli/efeitos dos fármacos , Expressão Gênica , Mutação , Fases de Leitura Aberta , Óperon , Peptídeos/imunologia , Peptídeos/farmacologia , Análise de Sequência de DNA , Staphylococcus aureus/metabolismo
14.
J Bacteriol ; 192(22): 5906-13, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20870773

RESUMO

Class IIa bacteriocins target a phylogenetically defined subgroup of mannose-phosphotransferase systems (man-PTS) on sensitive cells. By the use of man-PTS genes of the sensitive Listeria monocytogenes (mpt) and the nonsensitive Lactococcus lactis (ptn) species to rationally design a series of man-PTS chimeras and site-directed mutations, we identified an extracellular loop of the membrane-located protein MptC that was responsible for specific target recognition by the class IIa bacteriocins.


Assuntos
Antibiose , Proteínas de Bactérias/antagonistas & inibidores , Bacteriocinas/farmacologia , Lactococcus lactis/enzimologia , Listeria monocytogenes/enzimologia , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/antagonistas & inibidores , Sequência de Aminoácidos , Bacteriocinas/metabolismo , Lactococcus lactis/efeitos dos fármacos , Listeria monocytogenes/efeitos dos fármacos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Recombinação Genética , Alinhamento de Sequência
15.
Vet Microbiol ; 146(1-2): 124-31, 2010 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-20627619

RESUMO

Nukacin 3299 (formerly designated simulancin 3299), produced by a Staphylococcus simulans strain involved in bovine mastitis in Brazil, is the first peptide bacteriocin described in this staphylococcal species. With the intent to elucidate some aspects of its biology, nukacin 3299 was purified and characterized. The mass of the purified bacteriocin was shown to be 2957.3 Da, and the peptide N-terminal amino acids (KKKSGVI) were identified by Edman degradation. The nukacin 3299 structural gene, nukA, was detected by PCR and DNA sequencing, showing that this bacteriocin is identical to nukacin ISK-1, a 27-amino acid type-A (II) lantibiotic produced by Staphylococcus warneri ISK-1, isolated from a "nukadoko", in Japan. The genes involved in nukacin 3299 biosynthesis are located on plasmid pRJ97 (>27 kb). They have an organization similar to that of the nukacin ISK-1 gene cluster, excepted for the presence of an IS257/431 element (791 bp) present between the orf1 and nukA genes of the nukacin 3299 gene cluster. The presence of this insertion sequence is expected to affect the expression of orf1, whose function is presently unknown. Nukacin 3299 proved to be sensitive to proteolytic enzymes and relatively stable at different temperatures and between pH 3.0-9.0. Nukacin 3299 exhibited activity towards staphylococcal strains involved in bovine mastitis, showing a potential application on mastitis control, a disease with great economic impact.


Assuntos
Bacteriocinas/genética , Staphylococcus/metabolismo , Animais , Bacteriocinas/biossíntese , Bacteriocinas/isolamento & purificação , Sequência de Bases , Bovinos , Clonagem Molecular , Feminino , Genes Bacterianos/genética , Mastite Bovina/microbiologia , Dados de Sequência Molecular , Família Multigênica/genética , Reação em Cadeia da Polimerase/veterinária , Alinhamento de Sequência/veterinária , Análise de Sequência de DNA , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/veterinária
16.
Foodborne Pathog Dis ; 7(10): 1255-62, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20618078

RESUMO

In the present study, the bacteriocins produced by Staphylococcus aureus 4185, a strain isolated from bovine mastitis, were purified and partially characterized. After purification by ammonium sulfate precipitation, cation-exchange chromatography, and five runs of high-performance liquid chromatography (HPLC), antimicrobial activity was recovered with 40% and 80% isopropanol, suggesting that more than one antimicrobial peptide, named aureocins 4185, is produced by S. aureus 4185. Mass spectrometry analyses revealed three peptides eluted with 40% isopropanol: peptide A (2,305.3 +/-1.5 Da), peptide B (2,327.3 +/-1.5 Da), and peptide C (3,005.5 +/-1.5 Da), and two peptides eluted with 80% isopropanol: peptide D (6,413.5 +/-1.5 Da) and peptide E (12,834.5 +/-1.5 Da). Although five peptides have been detected, only four small peptide sequences were obtained by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF)/TOF mass spectrometry analyses: SLLEQFTGK (eluted with 40% isopropanol), ALLYDER, NNTSHNLPLGWFNVK, and NNLAQGTFNATK (eluted with 80% isopropanol). The sequences SLLEQFTGK and ALLYDER revealed identity with hypothetical peptides with unknown function. The sequences NNTSHNLPLGWFNVK and NNLAQGTFNATK showed similarity to a segment of a precursor of staphylococcal autolysins. The antimicrobial activity detected in the supernatant of strain 4185 proved to be resistant to heat treatment at 65°C; however, treatment at 80°C abolished completely its antimicrobial properties. The concentrated supernatant containing aureocins 4185 exhibited a strong bacteriolytic activity toward Micrococcus luteus ATCC 4698. Additionally, aureocins 4185 exhibited antagonistic activity against important foodborne pathogens, including Listeria monocytogenes, thus showing a potential application in food preservation.


Assuntos
Anti-Infecciosos/farmacologia , Bacteriocinas/farmacologia , Conservação de Alimentos/métodos , Staphylococcus aureus/metabolismo , Sequência de Aminoácidos , Animais , Bacillus cereus/efeitos dos fármacos , Bacteriocinas/química , Bacteriocinas/isolamento & purificação , Bovinos , Cromatografia Líquida de Alta Pressão , Estabilidade de Medicamentos , Temperatura Alta , Cinética , Listeria monocytogenes/efeitos dos fármacos , Mastite Bovina/microbiologia , Micrococcus luteus/efeitos dos fármacos , Dados de Sequência Molecular , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
17.
J Bacteriol ; 192(8): 2068-76, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20154137

RESUMO

The Abi protein family consists of putative membrane-bound metalloproteases. While they are involved in membrane anchoring of proteins in eukaryotes, little is known about their function in prokaryotes. In some known bacteriocin loci, Abi genes have been found downstream of bacteriocin structural genes (e.g., pln locus from Lactobacillus plantarum and sag locus from Streptococcus pyogenes), where they probably are involved in self-immunity. By modifying the profile hidden Markov model used to select Abi proteins in the Pfam protein family database, we show that this family is larger than presently recognized. Using bacteriocin-associated Abi genes as a means to search for novel bacteriocins in sequenced genomes, seven new bacteriocin-like loci were identified in Gram-positive bacteria. One such locus, from Lactobacillus sakei 23K, was selected for further experimental study, and it was confirmed that the bacteriocin-like genes (skkAB) exhibited antimicrobial activity when expressed in a heterologous host and that the associated Abi gene (skkI) conferred immunity against the cognate bacteriocin. Similar investigation of the Abi gene plnI and the Abi-like gene plnL from L. plantarum also confirmed their involvement in immunity to their cognate bacteriocins (PlnEF and PlnJK, respectively). Interestingly, the immunity genes from these three systems conferred a high degree of cross-immunity against each other's bacteriocins, suggesting the recognition of a common receptor. Site-directed mutagenesis demonstrated that the conserved motifs constituting the putative proteolytic active site of the Abi proteins are essential for the immunity function of SkkI, and to our knowledge, this represents a new concept in self-immunity.


Assuntos
Proteínas de Bactérias/metabolismo , Bacteriocinas/imunologia , Lactobacillus/imunologia , Lactobacillus/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Lactobacillus/genética , Mutagênese Sítio-Dirigida
18.
Probiotics Antimicrob Proteins ; 2(4): 233-40, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26781318

RESUMO

The aim of our study was to determine the genetic characterization and classification of Lb. gasseri K7 bacteriocins, comparison with bacteriocins of the Lb. gasseri LF221 strain and other related strains. Bacteriocin-encoding genes were amplified by PCR, subjected to DNA sequencing, and BLAST sequence analysis was performed to search the database for homologous peptides. Lb. gasseri K7 produces two two-peptide bacteriocins, named gassericin K7 A and gassericin K7 B. Their nucleotide sequences were deposited at GenBank, under accession numbers EF392861 for the gassericin K7 A and AY307382 for the gassericin K7 B. Analysis of gene clusters of bacteriocins in Lb. gasseri K7 strain revealed a 100 percent sequence identity with bacteriocins in LF221 strain. An active peptide of gassericin K7 B is homologous to the complementary peptide of gassericin T, and a complementary peptide of gassericin K7 B is homologous to the active peptide of gassericin T. Another surprising finding was that the sakacin T-beta peptide is partly homologous to the active peptide of gassericin K7 A, while the other sakacin T peptide (alfa) is partly homologous to the complementary peptide of gassericin K7 B. Gassericins of Lb. gasseri K7 strain were both classified as two-peptide bacteriocins. Human probiotic strains Lb. gasseri K7 and LF221 are different isolates but with identical bacteriocin genes. They produce wide-inhibitory spectra bacteriocins that are new members of two-peptide bacteriocins with some homologies to other bacteriocins in this group. Described bacteriocins offer a great potential in applications in food industry, pharmacy and biomedicine.

19.
Appl Environ Microbiol ; 74(11): 3615-7, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18424545

RESUMO

A beta-galactosidase reporter system for the analysis of promoter elements in Propionibacterium freudenreichii was designed. The pTD210 in vivo reporter vector was constructed using a promoterless lacZ gene from Bifidobacterium longum cloned into the pAMT1 plasmid. The utility of the pTD210 reporter vector was demonstrated by an investigation of six predicted promoters in P. freudenreichii. The system produced accurate and reproducible measurements that facilitated both promoter identification and the quantification of promoter activities.


Assuntos
Regulação Bacteriana da Expressão Gênica , Genes Reporter , Vetores Genéticos , Regiões Promotoras Genéticas , Propionibacterium/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Bifidobacterium/enzimologia , Bifidobacterium/genética , Clonagem Molecular , DNA Bacteriano/química , DNA Bacteriano/genética , Dados de Sequência Molecular , Plasmídeos , Análise de Sequência de DNA , beta-Galactosidase/biossíntese , beta-Galactosidase/genética
20.
Appl Environ Microbiol ; 73(23): 7542-7, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17933941

RESUMO

This report describes the first functional analysis of a bacteriocin immunity gene from Propionibacterium freudenreichii and its use as a selection marker for food-grade cloning. Cloning of the pcfI gene (previously orf5 [located as part of the pcfABC propionicin F operon]) rendered the sensitive host 1,000-fold more tolerant to the propionicin F bacteriocin. The physiochemical properties of the 127-residue large PcfI protein resemble those of membrane-bound immunity proteins from bacteriocin systems found in lactic acid bacteria. The high level of immunity conferred by pcfI allowed its use as a selection marker for plasmid transformation in P. freudenreichii. Electroporation of P. freudenreichii IFO12426 by use of the pcfI expression plasmid pSL102 and propionicin F selection (200 bacteriocin units/ml) yielded 10(7) transformants/microg DNA. The 2.7-kb P. freudenreichii food-grade cloning vector pSL104 consists of the pLME108 replicon, a multiple cloning site, and pcfI expressed from the constitutive P(pampS) promoter for selection. The pSL104 vector efficiently facilitated cloning of the propionicin T1 bacteriocin in P. freudenreichii. High-level propionicin T1 production (640 BU/ml) was obtained with the IFO12426 strain, and the food-grade propionicin T1 expression plasmid pSL106 was maintained by approximately 91% of the cells over 25 generations in the absence of selection. To the best of our knowledge this is the first report of an efficient cloning system that facilitates the generation of food-grade recombinant P. freudenreichii strains.


Assuntos
Bacteriocinas/genética , Clonagem Molecular/métodos , Propionibacterium/genética , Sequência de Aminoácidos , Sequência de Bases , Eletroporação , Microbiologia de Alimentos , Vetores Genéticos/genética , Dados de Sequência Molecular , Plasmídeos/genética , Transformação Bacteriana
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