Bam35 Tectivirus Intraviral Interaction Map Unveils New Function and Localization of Phage ORFan Proteins

ABSTRACT The family Tectiviridae comprises a group of tailless, icosahedral, membrane-containing bacteriophages that can be divided into two groups by their hosts, either Gram-negative or Gram-positive bacteria. While the first group is composed of PRD1 and nearly identical well-characterized lytic viruses, the second one includes more variable temperate phages, like GIL16 or Bam35, whose hosts are Bacillus cereus and related Gram-positive bacteria. In the genome of Bam35, nearly half of the 32 annotated open reading frames (ORFs) have no homologs in databases (ORFans), being putative proteins of unknown function, which hinders the understanding of their biology. With the aim of increasing knowledge about the viral proteome, we carried out a comprehensive yeast two-hybrid analysis of all the putative proteins encoded by the Bam35 genome. The resulting protein interactome comprised 76 unique interactions among 24 proteins, of which 12 have an unknown function. These results suggest that the P17 protein is the minor capsid protein of Bam35 and P24 is the penton protein, with the latter finding also being supported by iterative threading protein modeling. Moreover, the inner membrane transglycosylase protein P26 could have an additional structural role. We also detected interactions involving nonstructural proteins, such as the DNA-binding protein P1 and the genome terminal protein (P4), which was confirmed by coimmunoprecipitation of recombinant proteins. Altogether, our results provide a functional view of the Bam35 viral proteome, with a focus on the composition and organization of the viral particle. IMPORTANCE Tailless viruses of the family Tectiviridae can infect commensal and pathogenic Gram-positive and Gram-negative bacteria. Moreover, they have been proposed to be at the evolutionary origin of several groups of large eukaryotic DNA viruses and self-replicating plasmids. However, due to their ancient origin and complex diversity, many tectiviral proteins are ORFans of unknown function. Comprehensive protein-protein interaction (PPI) analysis of viral proteins can eventually disclose biological mechanisms and thus provide new insights into protein function unattainable by studying proteins one by one. Here we comprehensively describe intraviral PPIs among tectivirus Bam35 proteins determined using multivector yeast two-hybrid screening, and these PPIs were further supported by the results of coimmunoprecipitation assays and protein structural models. This approach allowed us to propose new functions for known proteins and hypothesize about the biological role of the localization of some viral ORFan proteins within the viral particle that will be helpful for understanding the biology of tectiviruses infecting Gram-positive bacteria.

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Recognition of Streptococcus pneumoniae and Muramyl Dipeptide by NOD2 Results in Potent Induction of MMP-9, Which Can Be Controlled by Lipopolysaccharide Stimulation

Infection and Immunity ◽

10.1128/iai.02150-14 ◽

2014 ◽

Vol 82 (12) ◽

pp. 4952-4958 ◽

Cited By ~ 10

Author(s):

Marloes Vissers ◽

Yvonne Hartman ◽

Laszlo Groh ◽

Dirk J. de Jong ◽

Marien I. de Jonge ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Mononuclear Cells ◽

Muramyl Dipeptide ◽

Tissue Inhibitor Of Metalloproteinase ◽

Gram Negative Bacteria ◽

Gram Positive ◽

Potent Inducer ◽

Gram Negative ◽

Content Type ◽

Gram Positive Bacteria

ABSTRACTMatrix metallopeptidase 9 (MMP-9) is a protease involved in the degradation of extracellular matrix collagen. Evidence suggests that MMP-9 is involved in pathogenesis duringStreptococcus pneumoniaeinfection. However, not much is known about the induction of MMP-9 and the regulatory processes involved. We show here that the Gram-positive bacteria used in this study induced large amounts of MMP-9, in contrast to the Gram-negative bacteria that were used. An important pathogen-associated molecular pattern (PAMP) for Gram-positive bacteria is muramyl dipeptide (MDP). MDP is a very potent inducer of MMP-9 and showed a dose-dependent MMP-9 induction. Experiments using peripheral blood mononuclear cells (PBMCs) from Crohn's disease patients with nonfunctional NOD2 showed that MMP-9 induction byStreptococcus pneumoniaeand MDP is NOD2 dependent. Increasing amounts of lipopolysaccharide (LPS), an important PAMP for Gram-negative bacteria, resulted in decreasing amounts of MMP-9. Moreover, the induction of MMP-9 by MDP could be counteracted by simultaneously adding LPS. The inhibition of MMP-9 expression by LPS was found to be regulated posttranscriptionally, independently of tissue inhibitor of metalloproteinase 1 (TIMP-1), an endogenous inhibitor of MMP-9. Collectively, these data show thatStreptococcus pneumoniaeis able to induce large amounts of MMP-9. These high MMP-9 levels are potentially involved inStreptococcus pneumoniaepathogenesis.

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Distinctive Binding of Avibactam to Penicillin-Binding Proteins of Gram-Negative and Gram-Positive Bacteria

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02102-15 ◽

2015 ◽

Vol 60 (2) ◽

pp. 752-756 ◽

Cited By ~ 34

Author(s):

Abdelhamid Asli ◽

Eric Brouillette ◽

Kevin M. Krause ◽

Wright W. Nichols ◽

François Malouin

Keyword(s):

Binding Proteins ◽

New Combination ◽

Gram Negative Bacteria ◽

Gram Positive ◽

Gram Negative ◽

Content Type ◽

Penicillin Binding Proteins ◽

Gram Positive Bacteria ◽

Fixed Concentration ◽

Competition Assays

ABSTRACTAvibactam is a novel non-β-lactam β-lactamase inhibitor that covalently acylates a variety of β-lactamases, causing inhibition. Although avibactam presents limited antibacterial activity, its acylation ability toward bacterial penicillin-binding proteins (PBPs) was investigated.Staphylococcus aureuswas of particular interest due to the reported β-lactamase activity of PBP4. The binding of avibactam to PBPs was measured by adding increasing concentrations to membrane preparations of a variety of Gram-positive and Gram-negative bacteria prior to addition of the fluorescent reagent Bocillin FL. Relative binding (measured here as the 50% inhibitory concentration [IC50]) to PBPs was estimated by quantification of fluorescence after gel electrophoresis. Avibactam was found to selectively bind to some PBPs. InEscherichia coli,Pseudomonas aeruginosa,Haemophilus influenzae, andS. aureus, avibactam primarily bound to PBP2, with IC50s of 0.92, 1.1, 3.0, and 51 μg/ml, respectively, whereas binding to PBP3 was observed inStreptococcus pneumoniae(IC50, 8.1 μg/ml). Interestingly, avibactam was able to significantly enhance labeling ofS. aureusPBP4 by Bocillin FL. In PBP competition assays withS. aureus, where avibactam was used at a fixed concentration in combination with varied amounts of ceftazidime, the apparent IC50of ceftazidime was found to be very similar to that determined for ceftazidime when used alone. In conclusion, avibactam is able to covalently bind to some bacterial PBPs. Identification of those PBP targets may allow the development of new diazabicyclooctane derivatives with improved affinity for PBPs or new combination therapies that act on multiple PBP targets.

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Effect of Lactose Monolaurate on Pathogenic and Nonpathogenic Bacteria

Applied and Environmental Microbiology ◽

10.1128/aem.07701-11 ◽

2012 ◽

Vol 78 (9) ◽

pp. 3465-3468 ◽

Cited By ~ 22

Author(s):

Ashwini Wagh ◽

Shujie Shen ◽

Fen Ann Shen ◽

Charles D. Miller ◽

Marie K. Walsh

Keyword(s):

Listeria Monocytogenes ◽

Antimicrobial Activities ◽

Gram Negative Bacteria ◽

Gram Positive ◽

Gram Negative ◽

Content Type ◽

Gram Positive Bacteria ◽

Lactose Monolaurate

ABSTRACTThe antimicrobial activities of sucrose monolaurate and a novel ester, lactose monolaurate (LML), were tested. Gram-positive bacteria were more susceptible than Gram-negative bacteria to both esters. The minimal bactericidal concentrations of LML were 5 to 9.5 mM forListeria monocytogenesisolates and 0.2 to 2 mM forMycobacteriumisolates.

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Development of Methionyl-tRNA Synthetase Inhibitors as Antibiotics for Gram-Positive Bacterial Infections

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00999-17 ◽

2017 ◽

Vol 61 (11) ◽

Cited By ~ 8

Author(s):

Omeed Faghih ◽

Zhongsheng Zhang ◽

Ranae M. Ranade ◽

J. Robert Gillespie ◽

Sharon A. Creason ◽

...

Keyword(s):

Protein Synthesis ◽

Bacterial Species ◽

Bacterial Load ◽

Trna Synthetase ◽

Resistant Bacteria ◽

Gram Positive ◽

Gram Negative ◽

Content Type ◽

Gram Positive Bacteria ◽

Methionyl Trna Synthetase

ABSTRACT Antibiotic-resistant bacteria are widespread and pose a growing threat to human health. New antibiotics acting by novel mechanisms of action are needed to address this challenge. The bacterial methionyl-tRNA synthetase (MetRS) enzyme is essential for protein synthesis, and the type found in Gram-positive bacteria is substantially different from its counterpart found in the mammalian cytoplasm. Both previously published and new selective inhibitors were shown to be highly active against Gram-positive bacteria with MICs of ≤1.3 μg/ml against Staphylococcus, Enterococcus, and Streptococcus strains. Incorporation of radioactive precursors demonstrated that the mechanism of activity was due to the inhibition of protein synthesis. Little activity against Gram-negative bacteria was observed, consistent with the fact that Gram-negative bacterial species contain a different type of MetRS enzyme. The ratio of the MIC to the minimum bactericidal concentration (MBC) was consistent with a bacteriostatic mechanism. The level of protein binding of the compounds was high (>95%), and this translated to a substantial increase in MICs when the compounds were tested in the presence of serum. Despite this, the compounds were very active when they were tested in a Staphylococcus aureus murine thigh infection model. Compounds 1717 and 2144, given by oral gavage, resulted in 3- to 4-log decreases in the bacterial load compared to that in vehicle-treated mice, which was comparable to the results observed with the comparator drugs, vancomycin and linezolid. In summary, the research describes MetRS inhibitors with oral bioavailability that represent a class of compounds acting by a novel mechanism with excellent potential for clinical development.

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Chitosan Augments Photodynamic Inactivation of Gram-Positive and Gram-Negative Bacteria

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00550-10 ◽

2011 ◽

Vol 55 (5) ◽

pp. 1883-1890 ◽

Cited By ~ 41

Author(s):

Tsuimin Tsai ◽

Hsiung-Fei Chien ◽

Tze-Hsien Wang ◽

Ching-Tsan Huang ◽

Yaw-Bee Ker ◽

...

Keyword(s):

Staphylococcus Epidermidis ◽

Light Exposure ◽

Gram Negative Bacteria ◽

Photodynamic Inactivation ◽

Gram Positive ◽

Gram Negative ◽

Content Type ◽

Gram Positive Bacteria ◽

Microbial Infections ◽

Promising Treatment

ABSTRACTAntimicrobial photodynamic inactivation (PDI) was shown to be a promising treatment modality for microbial infections. This study explores the effect of chitosan, a polycationic biopolymer, in increasing the PDI efficacy against Gram-positive bacteria, includingStaphylococcus aureus,Staphylococcus epidermidis,Streptococcus pyogenes, and methicillin-resistantS. aureus(MRSA), as well as the Gram-negative bacteriaPseudomonas aeruginosaandAcinetobacter baumannii. Chitosan at <0.1% was included in the antibacterial process either by coincubation with hematoporphyrin (Hp) and subjection to light exposure to induce the PDI effect or by addition after PDI and further incubation for 30 min. Under conditions in which Hp-PDI killed the microbe on a 2- to 4-log scale, treatment with chitosan at concentrations of as low as 0.025% for a further 30 min completely eradicated the bacteria (which were originally at ∼108CFU/ml). Similar results were also found with toluidine blue O (TBO)-mediated PDI in planktonic and biofilm cells. However, without PDI treatment, chitosan alone did not exert significant antimicrobial activity with 30 min of incubation, suggesting that the potentiated effect of chitosan worked after the bacterial damage induced by PDI. Further studies indicated that the potentiated PDI effect of chitosan was related to the level of PDI damage and the deacetylation level of the chitosan. These results indicate that the combination of PDI and chitosan is quite promising for eradicating microbial infections.

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Identification of the Lyso-Form N-Acyl Intramolecular Transferase in Low-GC Firmicutes

Journal of Bacteriology ◽

10.1128/jb.00099-17 ◽

2017 ◽

Vol 199 (11) ◽

Cited By ~ 16

Author(s):

Krista M. Armbruster ◽

Timothy C. Meredith

Keyword(s):

Fatty Acid ◽

Cell Envelope ◽

Transmembrane Protein ◽

Physiological Role ◽

Gram Positive ◽

Gram Negative ◽

Content Type ◽

Gram Positive Bacteria ◽

Unknown Gene ◽

Gram Negative Organisms

ABSTRACT Bacterial lipoproteins are embedded in the cell membrane of both Gram-positive and Gram-negative bacteria, where they serve numerous functions central to cell envelope physiology. Lipoproteins are tethered to the membrane by an N-acyl-S-(mono/di)-acyl-glyceryl-cysteine anchor that is variously acylated depending on the genus. In several low-GC, Gram-positive firmicutes, a monoacyl-glyceryl-cysteine with an N-terminal fatty acid (known as the lyso form) has been reported, though how it is formed is unknown. Here, through an intergenic complementation rescue assay in Escherichia coli, we report the identification of a common orthologous transmembrane protein in both Enterococcus faecalis and Bacillus cereus that is capable of forming lyso-form lipoproteins. When deleted from the native host, lipoproteins remain diacylated with a free N terminus, as maturation to the N-acylated lyso form is abolished. Evidence is presented suggesting that the previously unknown gene product functions through a novel intramolecular transacylation mechanism, transferring a fatty acid from the diacylglycerol moiety to the α-amino group of the lipidated cysteine. As such, the discovered gene has been named lipoprotein intramolecular transacylase (lit), to differentiate it from the gene for the intermolecular N-acyltransferase (lnt) involved in triacyl lipoprotein biosynthesis in Gram-negative organisms. IMPORTANCE This study identifies a new enzyme, conserved among low-GC, Gram-positive bacteria, that is involved in bacterial lipoprotein biosynthesis and synthesizes lyso-form lipoproteins. Its discovery is an essential first step in determining the physiological role of N-terminal lipoprotein acylation in Gram-positive bacteria and how these modifications impact bacterial cell envelope function.

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Lipoproteins of Gram-Positive Bacteria: Key Players in the Immune Response and Virulence

Microbiology and Molecular Biology Reviews ◽

10.1128/mmbr.00028-16 ◽

2016 ◽

Vol 80 (3) ◽

pp. 891-903 ◽

Cited By ~ 74

Author(s):

Minh Thu Nguyen ◽

Friedrich Götz

Keyword(s):

Immune Response ◽

Gram Negative Bacteria ◽

Gram Positive ◽

Gram Negative ◽

Content Type ◽

Gram Positive Bacteria ◽

Key Players ◽

Toll Like Receptor 2 ◽

Number Of Publications

SUMMARYSince the discovery in 1973 of the first of the bacterial lipoproteins (Lpp) inEscherichia coli, Braun's lipoprotein, the ever-increasing number of publications indicates the importance of these proteins. Bacterial Lpp belong to the class of lipid-anchored proteins that in Gram-negative bacteria are anchored in both the cytoplasmic and outer membranes and in Gram-positive bacteria are anchored only in the cytoplasmic membrane. In contrast to the case for Gram-negative bacteria, in Gram-positive bacteria lipoprotein maturation and processing are not vital. Physiologically, Lpp play an important role in nutrient and ion acquisition, allowing particularly pathogenic species to better survive in the host. Bacterial Lpp are recognized by Toll-like receptor 2 (TLR2) of the innate immune system. The important role of Lpp in Gram-positive bacteria, particularly in the phylumFirmicutes, as key players in the immune response and pathogenicity has emerged only in recent years. In this review, we address the role of Lpp in signaling and modulating the immune response, in inflammation, and in pathogenicity. We also address the potential of Lpp as promising vaccine candidates.

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Oligopolyphenylenevinylene-Conjugated Oligoelectrolyte Membrane Insertion Molecules Selectively Disrupt Cell Envelopes of Gram-Positive Bacteria

Applied and Environmental Microbiology ◽

10.1128/aem.03355-14 ◽

2015 ◽

Vol 81 (6) ◽

pp. 1949-1958 ◽

Cited By ~ 23

Author(s):

Jamie Hinks ◽

Wee Han Poh ◽

Justin Jang Hann Chu ◽

Joachim Say Chye Loo ◽

Guillermo C. Bazan ◽

...

Keyword(s):

Strain Improvement ◽

Membrane Insertion ◽

Gram Positive ◽

Gram Negative ◽

Content Type ◽

Gram Positive Bacteria ◽

Membrane Perturbation ◽

Transmission Electron ◽

Cell Envelopes ◽

Selection Of

ABSTRACTThe modification of microbial membranes to achieve biotechnological strain improvement with exogenous small molecules, such as oligopolyphenylenevinylene-conjugated oligoelectrolyte (OPV-COE) membrane insertion molecules (MIMs), is an emerging biotechnological field. Little is known about the interactions of OPV-COEs with their target, the bacterial envelope. We studied the toxicity of three previously reported OPV-COEs with a selection of Gram-negative and Gram-positive organisms and demonstrated that Gram-positive bacteria are more sensitive to OPV-COEs than Gram-negative bacteria. Transmission electron microscopy demonstrated that these MIMs disrupt microbial membranes and that this occurred to a much greater degree in Gram-positive organisms. We used a number of mutants to probe the nature of MIM interactions with the microbial envelope but were unable to align the membrane perturbation effects of these compounds to previously reported membrane disruption mechanisms of, for example, cationic antimicrobial peptides. Instead, the data support the notion that OPV-COEs disrupt microbial membranes through a suspected interaction with diphosphatidylglycerol (DPG), a major component of Gram-positive membranes. The integrity of model membranes containing elevated amounts of DPG was disrupted to a greater extent by MIMs than those prepared fromEscherichia colitotal lipid extracts alone.

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A Novel Mobilizing Tool Based on the Conjugative Transfer System of the IncM Plasmid pCTX-M3

Applied and Environmental Microbiology ◽

10.1128/aem.01205-20 ◽

2020 ◽

Vol 86 (17) ◽

Author(s):

Michał Dmowski ◽

Izabela Kern-Zdanowicz

Keyword(s):

Bacillus Subtilis ◽

Lactococcus Lactis ◽

Conjugative Transfer ◽

Conjugation System ◽

Gram Positive ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Gram Positive Bacteria ◽

Mobilizable Plasmid

ABSTRACT Conjugative plasmids are the main players in horizontal gene transfer in Gram-negative bacteria. DNA transfer tools constructed on the basis of such plasmids enable gene manipulation even in strains of clinical or environmental origin, which are often difficult to work with. The conjugation system of the IncM plasmid pCTX-M3 isolated from a clinical strain of Citrobacter freundii has been shown to enable efficient mobilization of oriTpCTX-M3-bearing plasmids into a broad range of hosts comprising Alpha-, Beta-, and Gammaproteobacteria. We constructed a helper plasmid, pMOBS, mediating such mobilization with an efficiency up to 1,000-fold higher than that achieved with native pCTX-M3. We also constructed Escherichia coli donor strains with chromosome-integrated conjugative transfer genes: S14 and S15, devoid of one putative regulator (orf35) of the pCTX-M3 tra genes, and S25 and S26, devoid of two putative regulators (orf35 and orf36) of the pCTX-M3 tra genes. Strains S14 and S15 and strains S25 and S26 are, respectively, up to 100 and 1,000 times more efficient in mobilization than pCTX-M3. Moreover, they also enable plasmid mobilization into the Gram-positive bacteria Bacillus subtilis and Lactococcus lactis. Additionally, the constructed E. coli strains carried no antibiotic resistance genes that are present in pCTX-M3 to facilitate manipulations with antibiotic-resistant recipient strains, such as those of clinical origin. To demonstrate possible application of the constructed tool, an antibacterial conjugation-based system was designed. Strain S26 was used for introduction of a mobilizable plasmid coding for a toxin, resulting in the elimination of over 90% of recipient E. coli cells. IMPORTANCE The conjugation of donor and recipient bacterial cells resulting in conjugative transfer of mobilizable plasmids is the preferred method enabling the introduction of DNA into strains for which other transfer methods are difficult to establish (e.g., clinical strains). We have constructed E. coli strains carrying the conjugation system of the IncM plasmid pCTX-M3 integrated into the chromosome. To increase the mobilization efficiency up to 1,000-fold, two putative regulators of this system, orf35 and orf36, were disabled. The constructed strains broaden the repertoire of tools for the introduction of DNA into the Gram-negative Alpha-, Beta-, and Gammaproteobacteria, as well as into Gram-positive bacteria such as Bacillus subtilis and Lactococcus lactis. The antibacterial procedure based on conjugation with the use of the orf35- and orf36-deficient strain lowered the recipient cell number by over 90% owing to the mobilizable plasmid-encoded toxin.

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The Fst/Ldr Family of Type I TA System Toxins: Potential Roles in Stress Response, Metabolism and Pathogenesis

Toxins ◽

10.3390/toxins12080474 ◽

2020 ◽

Vol 12 (8) ◽

pp. 474

Author(s):

Keith Weaver

Keyword(s):

Sugar Metabolism ◽

Type I ◽

Gram Positive ◽

Gram Negative ◽

Over Expression ◽

Gram Positive Bacteria ◽

The Family ◽

Peptide Toxins ◽

And Function

The parpAD1 locus was the first type I toxin–antitoxin (TA) system described in Gram-positive bacteria and was later determined to be the founding member of a widely distributed family of plasmid- and chromosomally encoded TA systems. Indeed, homology searches revealed that the toxin component, FstpAD1, is a member of the Fst/Ldr superfamily of peptide toxins found in both Gram-positive and Gram-negative bacteria. Regulation of the Fst and Ldr toxins is distinct in their respective Gram-positive and Gram-negative hosts, but the effects of ectopic over-expression are similar. While, the plasmid versions of these systems appear to play the canonical role of post-segregational killing stability mechanisms, the function of the chromosomal systems remains largely obscure. At least one member of the family has been suggested to play a role in pathogenesis in Staphylococcus aureus, while the regulation of several others appear to be tightly integrated with genes involved in sugar metabolism. After a brief discussion of the regulation and function of the foundational parpAD1 locus, this review will focus on the current information available on potential roles of the chromosomal homologs.

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