Journal of Bacteriology最新文献

Staphylococcus aureus is a major opportunistic pathogen in humans and animals. More than 90% of human nasal S. aureus isolates carry Sa3int-phages that integrate into the bacterial hlb gene coding for a sphingomyelinase. Sa3int-phages encode highly human-specific virulence factors that enable S. aureus to adapt to the human host. Thus, balancing mechanisms are necessary for the phage-bacteria coexistence. However, the factors that coordinate these interactions have yet to be discovered. Here, we elucidate the impact of the DNA-binding protein SarA on the life cycle of two prototypic S. aureus phages, Sa3int Φ13 and Sa5int Φ11. SarA promotes the propagation of both phages, albeit via different mechanisms. SarA promotes Φ11 propagation by repressing the glycosyltransferase TarM, which affects the glycosylation pattern of the phage receptor, wall teichoic acid, thereby improving phage adsorption. SarA also dampens the DNA damage response as indicated by the downregulation of the ci and mor phage promoters and the umuC SOS target gene, as well as inhibition of Φ11 inducibility. For Φ13, however, SarA promotes phage replication rather than inhibiting phage induction. The replication-deficient phage Φ13K-rep was SarA-insensitive, and phage gene expression was unaltered in the sarA mutant. These results highlight SarA as a regulator of temperate phage propagation and support its role as a DNA structural protein that promotes phage replication.

Importance: The dynamic gain and loss of temperate phages is crucial for bacteria to adapt to specific niches. In Staphylococcus aureus Sa3int, phages are highly prevalent in human strains but are missing in most animal strains. The mechanisms that balance phage-bacteria coexistence are only partially understood. We demonstrate that the DNA-binding protein SarA is a key regulator of the phage life cycle. SarA protects bacteria from phage induction in response to DNA damage, yet it can also promote phage propagation by altering the phage receptor or interfering with phage replication. SarA likely functions not only as a transcriptional factor, but also as a bacterial chromosome structural component that controls the phage life cycle at different levels.

Enterococcus faecium is a gram-positive bacterium that is resident to the intestines of animals including humans. E. faecium is also an opportunistic pathogen that causes multidrug-resistant (MDR) infections. Bacteriophages (phages) have been proposed as therapeutics for the treatment of MDR infections; however, an obstacle for phage therapy is the emergence of phage resistance. Despite this, the development of phage resistance can impact bacterial fitness. Thus, understanding the molecular basis of fitness costs associated with phage resistance can likely be leveraged as an antimicrobial strategy. We discovered that phage-resistant E. faecium harbor mutations in the cell wall hydrolase gene sagA. SagA cleaves crosslinked peptidoglycan (PG) involved in PG remodeling. We show that mutations in sagA compromised E. faecium PG hydrolysis. One sagA mutant, with a defect in cell envelope integrity, increased cellular permeability, and aberrant distribution of penicillin-binding proteins, was also more sensitive to β-lactam antibiotics. These changes correspond to a growth defect where cells have abnormal division septa, membrane blebbing, and aberrant cell shape. The dysregulation of the cell envelope caused by the sagA mutation alters the binding of phages to the E. faecium cell surface, where phage infection of E. faecium requires phages to localize to sites of peptidoglycan remodeling. Our findings show that by altering the function of a single PG hydrolase, E. faecium loses intrinsic β-lactam resistance. This indicates that phage therapy could help revive certain antibiotics when used in combination.IMPORTANCEEnterococcus faecium causes hospital-acquired infections and is frequently resistant to frontline antibiotics, including those that target the cell wall. Bacteriophages represent a promising alternative to combat such infections. However, bacterial adaptation to phage predation often results in resistance. Such resistance is frequently accompanied by fitness trade-offs, most notably altered antibiotic susceptibility. This study provides mechanistic insights into phage resistance-associated antibiotic sensitivity in E. faecium. We show that phage-resistant E. faecium carrying a mutation in the peptidoglycan hydrolase SagA has compromised cell envelope integrity, mislocalized penicillin-binding proteins, and become sensitized to β-lactam antibiotics. These findings highlight the potential of reviving antibiotics when used in combination with phages in the clinical setting.

Pseudomonas aeruginosa and Staphylococcus aureus are primary bacterial pathogens frequently isolated from the airways of cystic fibrosis patients. P. aeruginosa produces secondary metabolites that negatively impact the fitness of S. aureus, allowing P. aeruginosa to become the most prominent bacterium when the species are co-cultured. Some of these metabolites inhibit S. aureus respiration. SrrAB is a staphylococcal two-component regulatory system (TCRS) that responds to alterations in respiratory status to help S. aureus transition between fermentative and respiratory metabolisms. Using P. aeruginosa mutant strains and chemical genetics, we established that P. aeruginosa secondary metabolites, 2-heptyl-4-quinolone N-oxide (HQNO) in particular, inhibit S. aureus respiration, resulting in decreased SrrAB transcriptional output. Metabolomic analyses demonstrated that the ratio of NAD⁺ to NADH increased upon prolonged culture with HQNO. Consistent with this, the activity of the Rex transcriptional regulator, which senses and responds to alterations in the NAD⁺/NADH ratio, repressed srrAB promoter activity upon HQNO treatment. The presence of SrrAB increased fitness when cultured with HQNO and enhanced survival when challenged with P. aeruginosa. S. aureus strains with reduced ability to maintain redox homeostasis via fermentation had decreased fitness when challenged with HQNO and lower survival when challenged with P. aeruginosa. These findings led to a model wherein P. aeruginosa secreted HQNO inhibits S. aureus respiration, resulting in manipulation of the redox status in both the membrane and cytoplasm, altering the transcriptional activities of SrrAB and Rex, which promote fitness and survival by increasing carbon flux through fermentative pathways to maintain redox homeostasis.

Importance: Cystic fibrosis is a hereditary respiratory disease that predisposes patients to bacterial infections, often caused by Staphylococcus aureus and Pseudomonas aeruginosa. Secondary metabolites excreted by P. aeruginosa decrease S. aureus fitness during co-infection, ultimately eliminating it. The regulatory systems and mechanisms that S. aureus uses to detect and respond to these metabolites are unknown. The data presented demonstrate that two regulatory systems that are stimulated by alterations in membrane or cytosolic redox status respond to the P. aeruginosa-produced respiratory toxin 2-heptyl-4-quinolone N-oxide (HQNO) by increasing transcription of genes utilized for fermentation, thereby promoting fitness. This study describes interactions between these two bacterial pathogens that could be exploited to decrease pathogen burden in individuals living with cystic fibrosis.

During phototrophic growth, Cereibacter sphaeroides can use several carbon substrates that are central carbon intermediates (e.g., succinate and L-malate) or that require only a few steps to enter central carbon metabolism (e.g., acetate and D-malate). In addition, with light as the energy source, the carbon substrate provided will function as a carbon source for cell carbon synthesis only. Therefore, C. sphaeroides is ideally suited to understand the changes necessary to switch between different carbon sources and, consequently, to redirect carbon flow in central carbon metabolism. This study describes C. sphaeroides transposon mutants that have lost the ability to use one or more of the organic carbon sources 3-hydroxypropionate, acetate, L-malate, propionate/HCO₃^-, butyrate/HCO₃, L-lactate, D-lactate, D-malate, and L-glutamate. Pyruvate carboxylase and pyruvate dehydrogenase were confirmed to connect the precursor metabolite pools of pyruvate and oxaloacetate or acetyl-CoA, respectively, as was the ethylmalonyl-CoA pathway connecting acetyl-CoA and oxaloacetate pools. Transposon and in-frame deletion mutants suggest that 3-hydroxypropionate is oxidized to CO₂ and acetyl-CoA, involving a malonate semialdehyde dehydrogenase. The presence of this oxidative route makes pyruvate dehydrogenase dispensable during 3-hydroxypropionatedependent growth. Therefore, acetyl-CoA represents a second entry point into central carbon metabolism for 3-hydroxypropionate besides succinyl-CoA, and it is proposed that the simultaneous functioning of the two routes minimizes transiently produced CO₂/HCO₃^-. Another significant outcome of this study is the identification of genes encoding a L-glutamate TRipartite ATP-independent transporter, which was characterized biochemically 30 years ago.IMPORTANCESeveral aspects of the process of carbon assimilation, defined as the conversion of a carbon source into cell carbon, are conserved throughout life. For example, common building blocks give rise to proteins and nucleic acids, and the carbon for building blocks, cofactors, and secondary metabolites is derived from common precursor metabolites such as acetyl-CoA, pyruvate, or oxaloacetate. Using carbon substrates that require only one or a few steps to enter central carbon metabolism facilitates insights into the changes that occur to accommodate growth with different carbon substrates. In this study, transposon mutants that affect carbon flow in the core metabolism of Cereibacter sphaeroides were identified. Apparent redundancies of pathways can be explained by the need to maintain overall redox balance.

Nontuberculous mycobacteria (NTM) can form biofilms during human infection and in household plumbing systems, so understanding biofilm regulation could help us better treat and prevent NTM infections. Glucose drives NTM aggregation in vitro, and ammonium inhibits it, but the regulatory systems controlling this early step in biofilm formation are not understood. Here, in the model NTM Mycobacterium smegmatis, we show that multiple carbon and nitrogen sources have similar impacts on aggregation as glucose and ammonium , suggesting that the response to these nutrients is general and likely sensed through downstream, integrated signals. Next, we performed a transposon screen in M. smegmatis to uncover these putative regulatory nodes. Our screen revealed that mutating specific genes in the purine and pyrimidine biosynthesis pathways caused an aggregation defect, but supplementing with adenosine and guanosine had no impact on aggregation either in a purF mutant or WT. Realizing that the only genes we hit in purine or pyrimidine biosynthesis were those that utilized glutamine as a nitrogen donor, we pivoted to the hypothesis that intracellular glutamine could be a nitrogen-responsive node affecting aggregation. We tested this hypothesis in a defined M63 medium using targeted mass spectrometry. Indeed, intracellular glutamine increased with nitrogen availability and correlated with planktonic growth. Furthermore, a garA mutant, which has an artificially expanded glutamine pool in the growth phase, grew solely as planktonic cells even without nitrogen supplementation. Altogether, these results establish that intracellular glutamine controls M. smegmatis aggregation, and they introduce flux-dependent sensors as key components of the NTM biofilm regulatory system.IMPORTANCEA subset of nontuberculous mycobacteria (NTM), including Mycobacterium abscessus, are opportunistic pathogens that can cause severe pulmonary infections. Biofilm formation renders M. abscessus more tolerant to antibiotics; hence, the ability to inhibit NTM biofilm formation could help us better prevent and treat NTM infections. However, the regulatory systems controlling NTM biofilm formation, which could include targets for anti-biofilm therapeutics, are poorly understood. The significance of this work is that it reveals intracellular glutamine as an important node controlling the initiation of biofilm formation in the model NTM Mycobacterium smegmatis. Building on this foundation, future studies will investigate how NTM biofilms can be dispersed by altering glutamine levels and will describe how NTM translates intracellular glutamine to the alteration of surface adhesins.

The dihydroxyacetone phosphate (DHAP) shunt is a multifunctional pathway for the metabolism of 5'-deoxynucleosides and 5-deoxypentose sugars, such as 5'-methylthioadenosine (MTA) and 5'-deoxyadenosine (5dAdo), into DHAP and an aldehyde species depending on the substrate. Previous work revealed that Escherichia coli strains with the DHAP shunt can utilize exogenous MTA, 5dAdo, and derivatives thereof as sole carbon and energy sources for growth. However, if and how the DHAP shunt was regulated for 5'-deoxynucleoside and 5-deoxypentose sugar metabolism remained unknown. In the present work, the DHAP shunt genes (mtnK, mtnA, and ald2) and a putative transporter gene of E. coli ATCC 25922 are observed to form an operon, which can be expressed from two separate transcription start sites (TSSs). The distal, low-activity TSS appears to be constitutive, while the proximal primary TSS is regulated based on the identity of available growth substrates by at least two transcriptional regulators. First, YjhU, a deoxyribonucleoside operon repressor family regulator previously of unknown function that we designate as MtnR, functions as a repressor of the DHAP shunt operon when DHAP shunt substrates are absent. Further, the cyclic AMP receptor protein imposes carbon catabolite repression while glucose is available. Based on comparative sequence analysis, the E. coli DHAP shunt promoter region is highly conserved, including strains of the globally disseminated ST131 lineage of extraintestinal pathogenic E. coli, indicating a similar regulatory paradigm. Thus, the E. coli DHAP shunt is a previously unrecognized pathway for the use of 5'-deoxynucleosides and 5-deoxypentose sugars as alternative carbon sources when glucose is scarce.IMPORTANCEWhile not found in all Escherichia coli strains, the dihydroxyacetone phosphate (DHAP) shunt pathway is present in multiple lineages of extraintestinal pathogenic E. coli. The DHAP shunt allows E. coli strains to use a range of 5'-deoxynucleosides and 5-deoxypentose sugars as carbon and energy sources. These metabolites were previously considered waste products of cellular metabolism. This study identifies two transcriptional regulators that regulate the DHAP shunt operon, only allowing full expression when a DHAP shunt substrate is present and when glucose, a more-preferred carbon substrate, is absent. This demonstrates that the DHAP shunt is a genuine carbon metabolism pathway in E. coli and is placed under the hierarchy of carbon catabolite repression.

The metal cations of the first transition period fill up their 3d orbitals from 3d⁵ for Mn(II) to 3d¹⁰ for Zn(II). Enzymes use these cations as cofactors and exploit their individual chemical features for important catalytic reactions. A prerequisite for this process is metalation of the respective enzyme with the correct cation to form metal complexes, despite the presence of other competing transition metal cations. The first step to avoid mis-metalation requires maintenance of cytoplasmic cation homeostasis, which adjusts not only the concentration of an individual cation but also that of the overall metal-ion pools. This is achieved via a flow equilibrium of metal cation uptake by importers with broad substrate specificity combined with export of unwanted cations by efflux systems. A third group of cation importers with high substrate affinity contributes under metal starvation conditions. Experimental evidence for the existence of such a flow equilibrium comes from studies using the metal-resistant beta-proteobacterium Cupriavidus metallidurans. Central to the calibration of the pool of an individual metal cation are the regulators that control expression of the genes for the import and export pumps. A theoretical model that deduces how metal-cation discrimination may be performed by the respective regulator and the pathway from uptake of an external cation to correct metalation provides new insight into these processes.

Staphylococcus aureus and Pseudomonas aeruginosa are the two pathogens that colonize the airway of cystic fibrosis patients. As patients age, P. aeruginosa outcompetes S. aureus to become the predominant organism in the airway, which overlaps with worsening symptoms. This inverse correlation is partly due to the ability of P. aeruginosa to secrete secondary metabolites and virulence factors that are antagonistic to the host cells and other bacteria present. Several of these secondary metabolites inhibit S. aureus respiration. SaeRS is a two-component regulatory system that promotes the transcription of numerous virulence genes in S. aureus. The transcription of SaeRS-regulated genes is decreased as a function of respiratory status. The accumulation of intracellular fatty acids also negatively impacts the activity of SaeRS. Incubation of S. aureus with P. aeruginosa cell-free conditioned culture medium decreased the transcriptional output of the SaeRS system. Further analyses using P. aeruginosa mutant strains and chemical genetics determined that 2-heptyl-4-quinolone N-oxide (HQNO) was responsible for the SaeRS-dependent changes in gene regulation. Treatment with HQNO increased the abundance of cell-associated fatty acids. HQNO inhibits cell respiration, and the SaeRS system did not respond to HQNO treatment in a respiration-impaired S. aureus strain, which accumulates fatty acids. The data presented are consistent with a working model wherein treatment of S. aureus with HQNO inhibits respiration, increasing free fatty acid accumulation, which negatively impacts SaeRS signaling. This results in decreased expression of the SaeRS regulon, which has significant roles in pathogenesis.IMPORTANCEPseudomonas aeruginosa and Staphylococcus aureus are often co-isolated from the airways of cystic fibrosis patients. P. aeruginosa secretes non-essential metabolites that alter S. aureus physiology, providing P. aeruginosa with a competitive advantage. S. aureus can adapt to the presence of these metabolites, but the genetic mechanisms used to sense these P. aeruginosa-produced metabolites and/or the induced physiological changes are largely unknown. The S. aureus SaeRS two-component regulatory system positively regulates the expression of various virulence factors, including toxins and proteases, that facilitate adaptation to and survival in hostile host environments. This study demonstrates that the P. aeruginosa-produced respiratory toxin 2-heptyl-4-quinolone N-oxide inhibits respiration, decreasing the transcription of SaeRS-regulated genes and thereby decreasing virulence factor production. These findings could be exploited to decrease the ability of S. aureus to express virulence factors in various infection settings.

Individuals with poorly controlled diabetes mellitus often develop multispecies skin and soft tissue infections, with Staphylococcus aureus and Pseudomonas aeruginosa among the most prevalent bacteria isolated from infection sites worldwide. Diabetic infections are recalcitrant to conventional antibiotic regimens and may be a reservoir for emergent antibiotic-resistant bacterial strains. Supporting this, we have previously shown that rifampicin treatment elicits the emergence and expansion of rifampicin-resistant (Rif-r) S. aureus only in diabetic mice, potentially due to greater bacterial outgrowth increasing the frequency of resistance-conferring mutations. However, whether S. aureus exhibits altered resistance outcomes during multispecies diabetic infections is unclear. During co-infection with P. aeruginosa under normoglycemic conditions, S. aureus exhibits reduced growth and altered susceptibility to several antibiotics. In contrast, we previously observed that glucose availability allows S. aureus to largely overcome P. aeruginosa-mediated growth inhibition. Here, we explored S. aureus resistance outcomes under hyperglycemic conditions in the context of co-infection with P. aeruginosa during antibiotic challenge. We found that P. aeruginosa exoproducts regulated by the Pseudomonas quinolone signal quorum sensing system inhibit the emergence but not the expansion of Rif-r S. aureus in vitro under glucose-replete conditions. In contrast, we recovered equivalent Rif-r S. aureus burdens from diabetic mice during mono- and co-infection with P. aeruginosa. These results demonstrate that the diabetic infection microenvironment is conducive to emergent Rif-r S. aureus despite external pressures elicited by P. aeruginosa.IMPORTANCEPoorly controlled diabetes mellitus confers an increased susceptibility to bacterial infections, with Staphylococcus aureus and Pseudomonas aeruginosa frequently isolated from diabetic skin wounds. S. aureus readily develops antibiotic resistance during diabetic mono-infection under antibiotic pressure, but whether this occurs during diabetic co-infection is unclear. Under normoglycemic conditions, secreted P. aeruginosa factors alter S. aureus tolerance to several antibiotics. Here, we show that these P. aeruginosa exoproducts further inhibit the emergence of antibiotic-resistant S. aureus regardless of glucose availability in vitro, but this does not occur during subcutaneous co-infection in diabetic mice. These results provide initial insights regarding conditions that may inhibit S. aureus resistance development in hyperglycemic environments but underscore the influence of the host infection microenvironment in shaping resistance outcomes.

The aminoacyltransferase MurM is an important penicillin resistance determinant in Streptococcus pneumoniae. This enzyme attaches a serine or alanine to the side chain of lysine, the third residue of the pentapeptide of lipid II, resulting in branched muropeptides that can be crosslinked to stem peptides in peptidoglycan by penicillin binding proteins (PBPs). Deletion of murM results in only linear muropeptides, and more importantly, a significant reduction in resistance. Highly penicillin-resistant pneumococci express low-affinity PBPs, an altered MurM protein, and possess a highly branched cell wall. It has therefore been hypothesized that MurM, and thus branched muropeptides, are essential for resistance because they are better substrates for low-affinity PBPs. In this study, we found that neither the version of murM nor elevated levels of cell wall branching affected resistance levels. To further support this, we investigated whether branched muropeptide substrates compete better than linear versions with penicillin at the active site of low-affinity PBPs and quantified changes to the stem peptide composition of the resistant Pen6 strain in response to subinhibitory concentrations of penicillin. We found that the level of cell wall branching decreased during penicillin exposure. Together, our results do not support the idea that elevated levels of branched muropeptides (more active MurM) are important for either the function of low-affinity PBPs or the cell's response to penicillin. Nevertheless, since a functional MurM enzyme is important for resistance, we speculate that it might indirectly influence other functions related to cell wall synthesis and remodeling needed for a resistant phenotype.IMPORTANCEA fundamental understanding of the mechanisms behind antibiotic resistance is needed to find strategies to extend the clinical relevance of existing drugs. This study explores the relationship between cell wall composition and penicillin resistance in Streptococcus pneumoniae. Here, we confirm that branched peptide crosslinks in the cell wall are crucial for resistance but found no correlation between elevated branching levels and resistance. Our data suggest that the function of low-affinity penicillin binding proteins is not influenced by the lack of branched cell wall precursors. Instead, a branched cell wall might contribute to resistance via other cell wall biosynthesis and remodeling mechanisms. These insights could offer new perspectives on why a branched cell wall is important for penicillin resistance in pneumococci.