WhiA, a Protein of Unknown Function Conserved among Gram-Positive Bacteria, Is Essential for Sporulation inStreptomyces coelicolor A3(2)

ABSTRACT The whiA sporulation gene of Streptomyces coelicolor A3(2), which plays a key role in switching aerial hyphae away from continued extension growth and toward sporulation septation, was cloned by complementation of whiA mutants. DNA sequencing of the wild-type allele and five whiAmutations verified that whiA is a gene encoding a protein with homologues in all gram-positive bacteria whose genome sequence is known, whether of high or low G+C content. No function has been attributed to any of these WhiA-like proteins. In most cases, as inS. coelicolor, the whiA-like gene is downstream of other conserved genes in an operon-like cluster. Phenotypic analysis of a constructed disruption mutant confirmed that whiA is essential for sporulation. whiA is transcribed from at least two promoters, the most downstream of which is located within the preceding gene and is strongly up-regulated when colonies are undergoing sporulation. The up-regulation depends on a functionalwhiA gene, suggesting positive autoregulation, although it is not known whether this is direct or indirect. Unlike the promoters of some other sporulation-regulatory genes, the whiApromoter does not depend on the sporulation-specific ς factor encoded by whiG.

Download Full-text

Influence of the cell wall on ciprofloxacin susceptibility in selected wild-type Gram-negative and Gram-positive bacteria

International Journal of Antimicrobial Agents ◽

10.1016/j.ijantimicag.2003.12.015 ◽

2004 ◽

Vol 23 (6) ◽

pp. 627-630 ◽

Cited By ~ 18

Author(s):

Mercedes Berlanga ◽

M.Teresa Montero ◽

Jordi Hernández-Borrell ◽

Miquel Viñas

Keyword(s):

Cell Wall ◽

Wild Type ◽

Gram Positive ◽

Gram Negative ◽

Gram Positive Bacteria

Download Full-text

Inhibition of Methionyl-tRNA Synthetase by REP8839 and Effects of Resistance Mutations on Enzyme Activity

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00275-08 ◽

2008 ◽

Vol 53 (1) ◽

pp. 86-94 ◽

Cited By ~ 30

Author(s):

Louis S. Green ◽

James M. Bullard ◽

Wendy Ribble ◽

Frank Dean ◽

David F. Ayers ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Mechanistic Explanation ◽

Trna Synthetase ◽

Turnover Rates ◽

Wild Type ◽

Resistance Mutations ◽

Gram Positive ◽

Gram Positive Bacteria ◽

Human Orthologs ◽

Methionyl Trna Synthetase

ABSTRACT REP8839 is a selective inhibitor of methionyl-tRNA synthetase (MetRS) with antibacterial activity against a variety of gram-positive organisms. We determined REP8839 potency against Staphylococcus aureus MetRS and assessed its selectivity for bacterial versus human orthologs of MetRS. The inhibition constant (Ki ) of REP8839 was 10 pM for Staphylococcus aureus MetRS. Inhibition of MetRS by REP8839 was competitive with methionine and uncompetitive with ATP. Thus, high physiological ATP levels would actually facilitate optimal binding of the inhibitor. While many gram-positive bacteria, such as Staphylococcus aureus, express exclusively the MetRS1 subtype, many gram-negative bacteria express an alternative homolog called MetRS2. Some gram-positive bacteria, such as Streptococcus pneumoniae and Bacillus anthracis, express both MetRS1 and MetRS2. MetRS2 orthologs were considerably less susceptible to REP8839 inhibition. REP8839 inhibition of human mitochondrial MetRS was 1,000-fold weaker than inhibition of Staphylococcus aureus MetRS; inhibition of human cytoplasmic MetRS was not detectable, corresponding to >1,000,000-fold selectivity for the bacterial target relative to its cytoplasmic counterpart. Mutations in MetRS that confer reduced susceptibility to REP8839 were examined. The mutant MetRS enzymes generally exhibited substantially impaired catalytic activity, particularly in aminoacylation turnover rates. REP8839 Ki values ranged from 4- to 190,000-fold higher for the mutant enzymes than for wild-type MetRS. These observations provide a potential mechanistic explanation for the reduced growth fitness observed with MetRS mutant strains relative to that with wild-type Staphylococcus aureus.

Download Full-text

Role of Crotonyl Coenzyme A Reductase in Determining the Ratio of Polyketides Monensin A and Monensin B Produced byStreptomyces cinnamonensis

Journal of Bacteriology ◽

10.1128/jb.181.21.6806-6813.1999 ◽

1999 ◽

Vol 181 (21) ◽

pp. 6806-6813 ◽

Cited By ~ 33

Author(s):

Haibin Liu ◽

Kevin A. Reynolds

Keyword(s):

Coenzyme A ◽

Streptomyces Coelicolor ◽

Fermentation Medium ◽

Malt Extract ◽

Moderate Increase ◽

Wild Type ◽

Coding Region ◽

Gene Encoding ◽

Monensin A ◽

Coa Reductase

ABSTRACT The ccr gene, encoding crotonyl coenzyme A (CoA) reductase (CCR), was cloned from Streptomyces cinnamonensisC730.1 and shown to encode a protein with 90% amino acid sequence identity to the CCRs of Streptomyces collinus andStreptomyces coelicolor. A ccr-disrupted mutant, S. cinnamonensis L1, was constructed by inserting the hyg resistance gene into a unique BglII site within the ccr coding region. By use of theermE* promoter, the S. collinus ccr gene was expressed from plasmids in S. cinnamonensis C730.1/pHL18 and L1/pHL18. CCR activity in mutant L1 was shown to decrease by more than 90% in both yeast extract-malt extract (YEME) medium and a complex fermentation medium, compared to that in wild-type C730.1. Compared to C730.1, mutants C730.1/pHL18 and L1/pHL18 exhibited a huge increase in CCR activity (14- and 13-fold, respectively) in YEME medium and a moderate increase (3.7- and 2.7-fold, respectively) in the complex fermentation medium. In the complex fermentation medium,S. cinnamonensis L1 produced monensins A and B in a ratio of 12:88, dramatically lower than the 50:50 ratio observed for both C730.1 and C730.1/pHL18. Plasmid (pHL18)-based expression of theS. collinus ccr gene in mutant L1 increased the monensin A/monensin B ratio to 42:58. Labeling experiments with [1,2-13C2]acetate demonstrated the same levels of intact incorporation of this material into the butyrate-derived portion of monensin A in both C730.1 and mutant C730.1/pLH18 but a markedly decreased level of such incorporation in mutant L1. The addition of crotonic acid at 15 mM led to significant increases in the monensin A/monensin B ratio in C730.1 and C730.1/pHL18 but had no effect in S. cinnamonensis L1. These results demonstrate that CCR plays a significant role in providing butyryl-CoA for monensin A biosynthesis and is present in wild-type S. cinnamonensis C730.1 at a level sufficient that the availability of the appropriate substrate (crotonyl-CoA) is limiting.

Download Full-text

Branching of sporogenic aerial hyphae in sflA and sflB mutants of Streptomyces coelicolor correlates to ectopic localization of DivIVA and FtsZ in time and space

10.1101/2020.12.26.424426 ◽

2020 ◽

Author(s):

Le Zhang ◽

Joost Willemse ◽

Paula Yagüe ◽

Ellen de Waal ◽

Dennis Claessen ◽

...

Keyword(s):

Cell Division ◽

Streptomyces Coelicolor ◽

Constitutive Expression ◽

Hyphal Growth ◽

Lateral Wall ◽

Specific Cell ◽

Wild Type ◽

Aerial Hyphae ◽

In The Wild ◽

Z Ring

ABSTRACTBacterial cytokinesis starts with the polymerization of the tubulin-like FtsZ, which forms the cell division scaffold. SepF aligns FtsZ polymers and also acts as a membrane anchor for the Z-ring. While in most bacteria cell division takes place at midcell, during sporulation of Streptomyces many septa are laid down almost simultaneously in multinucleoid aerial hyphae. The genomes of streptomycetes encode two additional SepF paralogs, SflA and SflB, which can interact with SepF. Here we show that the sporogenic aerial hyphae of sflA and sflB mutants of Streptomyces coelicolor frequently branch, a phenomenon never seen in the wild-type strain. The branching coincided with ectopic localization of DivIVA along the lateral wall of sporulating aerial hyphae. Constitutive expression of SflA and SflB largely inhibited hyphal growth, further correlating SflAB activity to that of DivIVA. SflAB localized in foci prior to and after the time of sporulation-specific cell division, while SepF co-localized with active septum synthesis. Foci of FtsZ and DivIVA frequently persisted between adjacent spores in spore chains of sflA and sflB mutants, at sites occupied by SflAB in wild-type cells. This may be caused by the persistance of SepF multimers in the absence of SflAB. Taken together, our data show that SflA and SflB play an important role in the control of growth and cell division during Streptomyces development.

Download Full-text

NMR Hydrophilic Metabolomic Analysis of Bacterial Resistance Pathways using Multivalent Antimicrobials with Challenged and Unchallenged Wild Type and Mutated Gram-Positive Bacteria

International Journal of Molecular Sciences ◽

10.3390/ijms222413606 ◽

2021 ◽

Vol 22 (24) ◽

pp. 13606

Author(s):

Michelle L. Aries ◽

Mary J. Cloninger

Keyword(s):

Metabolic Pathways ◽

Qualitative Method ◽

Bacterial Resistance ◽

Proton Nuclear Magnetic Resonance ◽

Gram Negative Bacteria ◽

Wild Type ◽

Gram Positive ◽

Resistance Development ◽

Gram Positive Bacteria ◽

Positively Charged

Multivalent membrane disruptors are a relatively new antimicrobial scaffold that are difficult for bacteria to develop resistance to and can act on both Gram-positive and Gram-negative bacteria. Proton Nuclear Magnetic Resonance (1H NMR) metabolomics is an important method for studying resistance development in bacteria, since this is both a quantitative and qualitative method to study and identify phenotypes by changes in metabolic pathways. In this project, the metabolic differences between wild type Bacillus cereus (B. cereus) samples and B. cereus that was mutated through 33 growth cycles in a nonlethal dose of a multivalent antimicrobial agent were identified. For additional comparison, samples for analysis of the wild type and mutated strains of B. cereus were prepared in both challenged and unchallenged conditions. A C16-DABCO (1,4‑diazabicyclo-2,2,2-octane) and mannose functionalized poly(amidoamine) dendrimer (DABCOMD) were used as the multivalent quaternary ammonium antimicrobial for this hydrophilic metabolic analysis. Overall, the study reported here indicates that B. cereus likely change their peptidoglycan layer to protect themselves from the highly positively charged DABCOMD. This membrane fortification most likely leads to the slow growth curve of the mutated, and especially the challenged mutant samples. The association of these sample types with metabolites associated with energy expenditure is attributed to the increased energy required for the membrane fortifications to occur as well as to the decreased diffusion of nutrients across the mutated membrane.

Download Full-text

Apparent involvement of a multidrug transporter in the fluoroquinolone resistance of Streptococcus pneumoniae.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.41.6.1396 ◽

1997 ◽

Vol 41 (6) ◽

pp. 1396-1398 ◽

Cited By ~ 68

Author(s):

N N Baranova ◽

A A Neyfakh

Keyword(s):

Streptococcus Pneumoniae ◽

Ethidium Bromide ◽

Fluoroquinolone Resistance ◽

Cross Resistance ◽

Multidrug Transporter ◽

Wild Type ◽

Gram Positive ◽

Gram Positive Bacteria ◽

Energy Dependent

A Streptococcus pneumoniae strain selected for resistance to ethidium bromide demonstrated enhanced energy-dependent efflux of this toxic dye. Both the ethidium resistance and the ethidium efflux could be inhibited by the plant alkaloid reserpine. The ethidium-selected cells demonstrated cross-resistance to the fluoroquinolones norfloxacin and ciprofloxacin; this resistance could also be completely reversed by reserpine. Furthermore, reserpine potentiated the susceptibility of wild-type S. pneumoniae to fluoroquinolones and ethidium. The most plausible explanation for these results is that S. pneumoniae, like some other gram-positive bacteria, expresses a reserpine-sensitive multidrug transporter, which may play an important role in both intrinsic and acquired resistances of this pathogen to fluoroquinolone therapy.

Download Full-text

Alanyl-Phosphatidylglycerol and Lysyl-Phosphatidylglycerol Are Translocated by the Same MprF Flippases and Have Similar Capacities To Protect against the Antibiotic Daptomycin in Staphylococcus aureus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00370-12 ◽

2012 ◽

Vol 56 (7) ◽

pp. 3492-3497 ◽

Cited By ~ 39

Author(s):

Christoph J. Slavetinsky ◽

Andreas Peschel ◽

Christoph M. Ernst

Keyword(s):

Staphylococcus Aureus ◽

Deletion Mutant ◽

Wild Type ◽

Gram Positive ◽

Content Type ◽

Wild Type Level ◽

Bacterial Membranes ◽

Gram Positive Bacteria ◽

Cytoplasmic Membranes ◽

Simultaneous Expression

ABSTRACTThe lysinylation of negatively charged phosphatidylglycerol by MprF proteins reduces the affinity of cationic antimicrobial peptides (CAMPs) for bacterial cytoplasmic membranes and reduces the susceptibility of several Gram-positive bacterial pathogens to CAMPs. MprF ofStaphylococcus aureusencompasses a lysyl-phosphatidylglycerol (Lys-PG) synthase and a Lys-PG flippase domain. In contrast,Clostridium perfringensencodes two MprF homologs which specifically synthesize alanyl-phosphatidylglycerol (Ala-PG) or Lys-PG, while only the Lys-PG synthase is fused to a putative flippase domain. It remains unknown whether cationic Lys-PG and zwitterionic Ala-PG differ in their capacities to be translocated by MprF flippases and if both can reduce CAMP susceptibility in Gram-positive bacteria. By expressing the MprF proteins ofC. perfringensin anS. aureus mprFdeletion mutant, we found that both lipids can be efficiently produced inS. aureus. Simultaneous expression of the Lys-PG and Ala-PG synthases led to the production of both lipids and slightly increased the overall amounts of aminoacyl phospholipids. Ala-PG production by the correspondingC. perfringensenzyme did not affect susceptibility to CAMPs such as nisin and gallidermin or to the CAMP-like antibiotic daptomycin. However, coexpression of the Ala-PG synthase with flippase domains of Lys-PG synthesizing MprF proteins led to a wild-type level of daptomycin susceptibility, indicating that Ala-PG can also protect bacterial membranes against daptomycin and suggesting that Lys-PG flippases can also translocate the related lipid Ala-PG. Thus, bacterial aminoacyl phospholipid flippases exhibit more relaxed substrate specificity and Ala-PG and Lys-PG are more similar in their capacities to modulate membrane functions than anticipated.

Download Full-text

Enhancing Isoprene Production by Genetic Modification of the 1-Deoxy-d-Xylulose-5-Phosphate Pathway inBacillus subtilis

Applied and Environmental Microbiology ◽

10.1128/aem.02341-10 ◽

2011 ◽

Vol 77 (7) ◽

pp. 2399-2405 ◽

Cited By ~ 70

Author(s):

Junfeng Xue ◽

Birgitte K. Ahring

Keyword(s):

Sigma Factor ◽

Type Strain ◽

Wild Type Strain ◽

Stress Factors ◽

Alternative Sigma Factor ◽

Wild Type ◽

Gram Positive ◽

Gram Positive Bacteria ◽

Overexpression Strain ◽

As Stress

ABSTRACTTo enhance the production of isoprene, a volatile 5-carbon hydrocarbon, in the Gram-positive spore-forming rod-shaped bacteriumBacillus subtilis, 1-deoxy-d-xylulose-5-phosphate synthase (Dxs) and 1-deoxy-d-xylulose-5-phosphate reductoisomerase (Dxr) were overexpressed inB. subtilisDSM 10. For the strain that overexpresses Dxs, the yield of isoprene was increased 40% over that by the wild-type strain. In the Dxr overexpression strain, the level of isoprene production was unchanged. Overexpression of Dxr together with Dxs showed an isoprene production level similar to that of the Dxs overproduction strain. The effects of external factors, such as stress factors including heat (48°C), salt (0.3 M NaCl), ethanol (1%), and oxidative (0.005% H2O2) stress, on isoprene production were further examined. Heat, salt, and H2O2induced isoprene production; ethanol inhibited isoprene production. In addition, induction and repression effects are independent of SigB, which is the general stress-responsive alternative sigma factor of Gram-positive bacteria.

Download Full-text

Bacillus subtilis YhaM, a Member of a New Family of 3′-to-5′ Exonucleases in Gram-Positive Bacteria

Journal of Bacteriology ◽

10.1128/jb.184.22.6250-6259.2002 ◽

2002 ◽

Vol 184 (22) ◽

pp. 6250-6259 ◽

Cited By ~ 37

Author(s):

Irina A. Oussenko ◽

Roberto Sanchez ◽

David H. Bechhofer

Keyword(s):

Bacillus Subtilis ◽

Mrna Turnover ◽

Wild Type ◽

Rnase R ◽

Gram Positive ◽

Gram Positive Bacteria ◽

New Family ◽

Double Stranded Dna ◽

Ob Fold ◽

Related Proteins

ABSTRACT A strain of Bacillus subtilis lacking two 3′-to-5′ exoribonucleases, polynucleotide phosphorylase (PNPase) and RNase R, was used to purify another 3′-to-5′ exoribonuclease, which is encoded by the yhaM gene. YhaM was active in the presence of Mn2+ (or Co2+), was inactive in the presence of Mg2+, and could also degrade single-stranded DNA. The half-life of bulk mRNA in a mutant lacking PNPase, RNase R, and YhaM was not significantly different from that of the wild type, suggesting the existence of additional activities that can participate in mRNA turnover. Sequence homologues of YhaM were found only in gram-positive organisms. The Staphylococcus aureus homologue, CBF1, which had been characterized as a double-stranded DNA binding protein involved in plasmid replication, was also shown to be an Mn2+-dependent exoribonuclease. YhaM protein has a C-terminal “HD domain,” found in metal-dependent phosphohydrolases. By structure modeling, it was shown that YhaM also contains an N-terminal “OB-fold,” present in many oligosaccharide- and oligonucleotide-binding proteins. The combination of these two domains is unique. Thus, YhaM and 10 related proteins from gram-positive organisms constitute a new exonuclease family.

Download Full-text