Scrutinizing a Lactococcus lactis mutant with enhanced capacity for extracellular electron transfer reveals a unique role for a novel type-II NADH dehydrogenase.

Lactococcus lactis, a lactic acid bacterium used in food fermentations and commonly found in the human gut, is known to possess a fermentative metabolism. L. lactis, however, has been demonstrated to transfer metabolically generated electrons to external electron acceptors, a process termed extracellular electron transfer (EET). Here, we investigated an L. lactis mutant with an unusually high capacity for EET that was obtained in an adaptive laboratory evolution (ALE) experiment. First, we investigated how global gene expression had changed, and found that amino acid metabolism and nucleotide metabolism had been affected significantly. One of the most significantly upregulated genes encoded the NADH dehydrogenase NoxB. We found that this upregulation was due to a mutation in the promoter region of NoxB, which abolished carbon catabolite repression. A unique role of NoxB in EET could be attributed and it was directly verified, for the first time, that NoxB could support respiration in L. lactis. NoxB, was shown to be a novel type-II NADH dehydrogenase that is widely distributed among gut microorganisms. This work expands our understanding of EET in Gram-positive electroactive microorganisms and the special significance of a novel type-II NADH dehydrogenase in EET.IMPORTANCEElectroactive microorganisms with extracellular electron transfer (EET) ability play important roles in biotechnology and ecosystems. To date, there have been many investigations aiming at elucidating the mechanisms behind EET, and determining the relevance of EET for microorganisms in different niches. However, how EET can be enhanced and harnessed for biotechnological applications has been less explored. Here, we compare the transcriptomes of an EET-enhanced L. lactis mutant with its parent and elucidate the underlying reason for its superior performance. We find that one of the most significantly upregulated genes is the gene encoding the NADH dehydrogenase NoxB, and that upregulation is due to a mutation in the catabolite-responsive element that abolishes carbon catabolite repression. We demonstrate that NoxB has a special role in EET, and furthermore show that it supports respiration to oxygen, which has never been done previously. In addition, a search reveals that this novel NoxB-type NADH dehydrogenase is widely distributed among gut microorganisms.

StkP- and PhpP-Mediated Posttranslational Modifications Modulate the S. pneumoniae Metabolism, Polysaccharide Capsule, and Virulence.

Pneumococcal Ser/Thr kinase (StkP) and its cognate phosphatase (PhpP) play a crucial role in bacterial cytokinesis. However, their individual and reciprocal metabolic and virulence regulation-related functions have yet to be adequately investigated in encapsulated pneumococci. Here, we demonstrate that the encapsulated pneumococcal strain D39-derived D39ΔPhpP and D39ΔStkP mutants displayed differential cell division defects and growth patterns when grown in chemically defined media supplemented with glucose or nonglucose sugars as the sole carbon source. Microscopic and biochemical analyses supported by RNA-seq-based global transcriptomic analyses of these mutants revealed significantly down- and upregulated polysaccharide capsule formation and cps2 genes in D39ΔPhpP and D39ΔStkP mutants, respectively. While StkP and PhpP individually regulated several unique genes, they also participated in sharing the regulation of the same set of differentially regulated genes. Cps2 genes were reciprocally regulated in part by the StkP/PhpP-mediated reversible phosphorylation but independent of the MapZ-regulated cell division process. StkP-mediated dose-dependent phosphorylation of CcpA proportionately inhibited CcpA-binding to Pcps2A, supporting increased cps2 gene expression and capsule formation in D39ΔStkP. While the attenuation of the D39ΔPhpP mutant in two mouse infection models corroborated with several downregulated capsules-, virulence-, and phosphotransferase systems (PTS)-related genes, the D39ΔStkP mutant with increased amounts of polysaccharide capsules displayed significantly decreased virulence in mice compared to the D39 wild-type, but more virulence compared to D39ΔPhpP. NanoString technology-based inflammation-related gene expression and Meso Scale Discovery-based multiplex chemokine analysis of human lung cells cocultured with these mutants confirmed their distinct virulence phenotypes. StkP and PhpP may, therefore, serve as critical therapeutic targets.

luxS contributes to intramacrophage survival of Streptococcus agalactiae by positively affecting the expression of fruRKI operon.

The LuxS quorum sensing system is a widespread system employed by many bacteria for cell-to-cell communication. The luxS gene has been demonstrated to play a crucial role in intramacrophage survival of piscine Streptococcus agalactiae, but the underlying mechanism remains largely unknown. In this study, transcriptome analysis, followed by the luxS gene deletion and subsequent functional studies, confirmed that impaired bacterial survival inside macrophages due to the inactivation of luxS was associated with reduced transcription of the fruRKI operon, encoding the fructose-specific phosphotransferase system. Further, luxS was determined not to enhance the transcription of fruRKI operon by binding its promoter, but to upregulate the expression of this operon via affecting the binding ability of catabolite control protein A (CcpA) to the catabolite responsive element (cre) in the promoter of fruRKI. Collectively, our study identifies a novel and previously unappreciated role for luxS in bacterial intracellular survival, which may give a more thorough understanding of the immune evasion mechanism in S. agalactiae.

Effects of 2-carba-cyclic phosphatidic acid derivatives on IL-1ß-stimulated human chondrocytes.

Osteoarthritis (OA) is a common joint disease characterized by the breakdown of subchondral bone and cartilage damage, most often affecting middle-aged and elderly people. Although the etiology of OA is still unknown, some reports suggest that inflammatory factors such as interleukin (IL)- 1ß mediate the progression of OA. To investigate the effect of IL-1ß and the possibility of treatment for OA, we applied 2-carba-cyclic phosphatidic acid (2ccPA) and its derivatives on human chondrocytes. 2ccPA is a synthesized phospholipid derived from a bioactive phospholipid mediator: cyclic phosphatidic acid (cPA). It has been previously reported that 2ccPA exhibits anti-inflammatory and chondroprotective effects in an OA animal model. 2ccPA and its ring-opened body (ROB) derivative significantly suppressed IL-1ß-induced upregulation of IL-6, matrix metalloproteinase-13, and cyclooxygenase-2, as well as the degradation of type II collagen and aggrecan. However, the other two derivatives, namely the deacylated and ring-opened deacylated bodies, showed little effect on an IL-1ß-exposed human chondrosarcoma cell-line. These data suggest that the intactness of 2ccPA and ROB is essential for anti-inflammatory effects on OA. Collectively, this study provides evidence that 2ccPA and ROB would be novel therapeutic agents for OA.

Role of C-Reactive Protein and Erythrocyte Sedimentation Rate in the Diagnosis and Monitoring of Treatment Response in Treatment Naïve Subjects with Chronic Pulmonary Aspergillosis.

PURPOSE: The role of C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) for diagnosing chronic pulmonary aspergillosis (CPA) remains unknown. Herein, we investigate the diagnostic performance of serum ESR and CRP in CPA. METHODS: We retrospectively analyzed the data of treatment-naïve subjects with CPA and diseased controls (post-tuberculosis lung disease on CT thorax). We treated CPA subjects with six months of oral itraconazole. Our primary objective was to evaluate the sensitivity and specificity of ESR and CRP in diagnosing CPA. The key secondary objective was to study the change in the inflammatory markers with treatment. RESULTS: We included 434 subjects and 20 diseased controls. The sensitivity and specificity of ESR (n = 434) and CRP (at cut-off value of 10 mg/L, n = 308) in diagnosing CPA were 42.9% and 65%, and 52.3% and 65%, respectively. Both ESR and CRP had erratic trend following treatment. ESR and CRP declined or remained stable in approximately 60% of subjects but increased in approximately 40% of the subjects despite treatment. CONCLUSION: Serum CRP and ESR have limited utility in diagnosing and following subjects with CPA.

Characterization of Treatment Response Outcomes in Chronic Pulmonary Aspergillosis: CPAnet Definitions Versus the Existing Criteria.

BACKGROUND: The chronic pulmonary aspergillosis network (CPAnet) has recently proposed definitions for treatment outcomes in CPA. However, these definitions need to be validated. Herein, we evaluate the agreement between the existing and the CPAnet definitions for response assessment. METHODS: We enrolled consecutive treatment-naïve CPA subjects (between January 2021 and June 2021) who received six months of itraconazole therapy and followed them for an additional six months after treatment discontinuation. We retrospectively applied the CPAnet criteria and compared the agreement between the existing and the CPAnet criteria for response assessment (primary objective). We also assessed if adding weight loss (> 5% from baseline) as a component improved the performance of the CPAnet criteria. RESULTS: We included 43 (mean age, 47.4 years) CPA subjects. The existing and the CPAnet criteria categorized 29 (67.4%) and 30 (69.8%) subjects as treatment success, respectively, at treatment completion. There was substantial (kappa = 0.73; p < 0.0001) agreement between the two definitions. However, both criteria did not identify eight subjects requiring treatment re-initiation within three months. There was an increment in the sensitivity of both criteria (by 36%) for identifying treatment failure after incorporating ≥ 5% weight loss as an element of worsening. CONCLUSION: The CPAnet definitions correctly categorized treatment outcomes in most cases of CPA. The addition of weight change would further enhance the performance of the CPAnet treatment outcome definitions.

Astrocytic Neuroimmunological Roles Interacting with Microglial Cells in Neurodegenerative Diseases.

Both astrocytic and microglial functions have been extensively investigated in healthy subjects and neurodegenerative diseases. For astrocytes, not only various sub-types were identified but phagocytic activity was also clarified recently and is making dramatic progress. In this review paper, we mostly focus on the functional role of astrocytes in the extracellular matrix and on interactions between reactive astrocytes and reactive microglia in normal states and in neurodegenerative diseases, because the authors feel it is necessary to elucidate the mechanisms among activated glial cells in the pathology of neurological diseases in order to pave the way for drug discovery. Finally, we will review cyclic phosphatidic acid (cPA), a naturally occurring phospholipid mediator that induces a variety of biological activities in the brain both in vivo and in vitro. We propose that cPA may serve as a novel therapeutic molecule for the treatment of brain injury and neuroinflammation.

Staphylococcus aureus Does Not Synthesize Arginine from Proline under Physiological Conditions.

The Gram-positive pathogen Staphylococcus aureus is the only bacterium known to synthesize arginine from proline via the arginine-proline interconversion pathway despite having genes for the well-conserved glutamate pathway. Since the proline-arginine interconversion pathway is repressed by CcpA-mediated carbon catabolite repression (CCR), CCR has been attributed to the arginine auxotrophy of S. aureus. Using ribose as a secondary carbon source, here, we demonstrate that S. aureus arginine auxotrophy is not due to CCR but due to the inadequate concentration of proline degradation product. Proline is degraded by proline dehydrogenase (PutA) into pyrroline-5-carboxylate (P5C). Although the PutA expression was fully induced by ribose, the P5C concentration remained insufficient to support arginine synthesis because P5C was constantly consumed by the P5C reductase ProC. When the P5C concentration was artificially increased by either PutA overexpression or proC deletion, S. aureus could synthesize arginine from proline regardless of carbon source. In contrast, when the P5C concentration was reduced by overexpression of proC, it inhibited the growth of the ccpA deletion mutant without arginine. Intriguingly, the ectopic expression of the glutamate pathway enzymes converted S. aureus into arginine prototroph. In an animal experiment, the arginine-proline interconversion pathway was not required for the survival of S. aureus. Based on these results, we concluded that S. aureus does not synthesize arginine from proline under physiological conditions. We also propose that arginine auxotrophy of S. aureus is not due to the CcpA-mediated CCR but due to the inactivity of the conserved glutamate pathway. IMPORTANCE Staphylococcus aureus is a versatile Gram-positive human pathogen infecting various human organs. The bacterium's versatility is partly due to efficient metabolic regulation via the carbon catabolite repression system (CCR). S. aureus is known to interconvert proline and arginine, and CCR represses the synthesis of both amino acids. However, when CCR is released by a nonpreferred carbon source, S. aureus can synthesize proline but not arginine. In this study, we show that, in S. aureus, the intracellular concentration of pyrroline-5-carboxylate (P5C), the degradation product of proline and the substrate of proline synthesis, is too low to synthesize arginine from proline. These results call into question the notion that S. aureus synthesizes arginine from proline.

Glucose Mediates Niche-Specific Repression of Staphylococcus aureus Toxic Shock Syndrome Toxin-1 through the Activity of CcpA in the Vaginal Environment.

Staphylococcus aureus chronically colonizes up to 30% of the human population on the skin or mucous membranes, including the nasal tract or vaginal canal. While colonization is often benign, this bacterium also has the capability to cause serious infections. Menstrual toxic shock syndrome (mTSS) is a serious toxinosis associated with improper use of tampons, which can induce an environment that is favorable to the production of the superantigen known as toxic shock syndrome toxin-1 (TSST-1). To better understand environmental signaling that influences TSST-1 production, we analyzed expression in the prototype mTSS strain S. aureus MN8. Using transcriptional and protein-based analysis in two niche-related media, we observed that TSST-1 expression was significantly higher in synthetic nasal medium (SNM) than in vaginally defined medium (VDM). One major divergence in medium composition was high glucose concentration in VDM. The glucose-dependent virulence regulator gene ccpA was deleted in MN8, and, compared with wild-type MN8, we observed increased TSST-1 expression in the ΔccpA mutant when grown in VDM, suggesting that TSST-1 is repressed by catabolite control protein A (CcpA) in the vaginal environment. We were able to relieve CcpA-mediated repression by modifying the glucose level in vaginal conditions, confirming that changes in nutritional conditions contribute to the overexpression of TSST-1 that can lead to mTSS. We also compared CcpA-mediated repression to other key regulators of tst, finding that CcpA regulation is dominant compared to other characterized regulatory mechanisms. This study underlines the importance of environmental signaling for S. aureus pathogenesis in the context of mTSS. IMPORTANCE Menstrual toxic shock syndrome (mTSS) is caused by strains of Staphylococcus aureus that overproduce a toxin known as toxic shock syndrome toxin-1 (TSST-1). This work studied how glucose levels in a model vaginal environment could influence the amount of TSST-1 that is produced by S. aureus. We found that high levels of glucose repress TSST-1 production, and this is done by a regulatory protein called catabolite control protein A (CcpA). The research also demonstrated that, compared with other regulatory proteins, the CcpA regulator appears to be the most important for maintaining low levels of TSST-1 in the vaginal environment, and this information helps to understand how changes in the vaginal environmental can lead to mTSS.

Genome-wide analysis of in vivo CcpA binding with and without its key co-factor HPr in the major human pathogen group A Streptococcus.

Catabolite control protein A (CcpA) is a master regulator of carbon source utilization and contributes to the virulence of numerous medically important Gram-positive bacteria. Most functional assessments of CcpA, including interaction with its key co-factor HPr, have been performed in nonpathogenic bacteria. In this study we aimed to identify the in vivo DNA binding profile of CcpA and assess the extent to which HPr is required for CcpA-mediated regulation and DNA binding in the major human pathogen group A Streptococcus (GAS). Using a combination RNAseq/ChIP-seq approach, we found that CcpA affects transcript levels of 514 of 1667 GAS genes (31%) whereas direct DNA binding was identified for 105 GAS genes. Three of the directly regulated genes encode the key GAS virulence factors Streptolysin S, PrtS (IL-8 degrading proteinase), and SpeB (cysteine protease). Mutating CcpA Val301 to Ala (strain 2221-CcpA-V301A) abolished interaction between CcpA and HPr and impacted the transcript levels of 205 genes (40%) in the total CcpA regulon. By ChIP-seq analysis, CcpAV301A bound to DNA from 74% of genes bound by wild-type CcpA, but generally with lower affinity. These data delineate the direct CcpA regulon and clarify the HPr-dependent and independent activities of CcpA in a key pathogenic bacterium.

Proteome constraints reveal targets for improving microbial fitness in nutrient-rich environments.

Cells adapt to different conditions via gene expression that tunes metabolism for maximal fitness. Constraints on cellular proteome may limit such expression strategies and introduce trade-offs. Resource allocation under proteome constraints has explained regulatory strategies in bacteria. It is unclear, however, to what extent these constraints can predict evolutionary changes, especially for microorganisms that evolved under nutrient-rich conditions, i.e., multiple available nitrogen sources, such as Lactococcus lactis. Here, we present a proteome-constrained genome-scale metabolic model of L. lactis (pcLactis) to interpret growth on multiple nutrients. Through integration of proteomics and flux data, in glucose-limited chemostats, the model predicted glucose and arginine uptake as dominant constraints at low growth rates. Indeed, glucose and arginine catabolism were found upregulated in evolved mutants. At high growth rates, pcLactis correctly predicted the observed shutdown of arginine catabolism because limited proteome availability favored lactate for ATP production. Thus, our model-based analysis is able to identify and explain the proteome constraints that limit growth rate in nutrient-rich environments and thus form targets of fitness improvement.

Gene rppA co-regulated by LRR, SigA, and CcpA mediates antibiotic resistance in Bacillus thuringiensis.

Antibiotic resistance genes are usually tightly controlled by transcription factors and RNA regulatory elements including sRNAs, riboswitches, and attenuators, and their expression is activated to respond to antibiotic exposure. In previous work, we revealed that the rppA gene is regulated by attenuator LRR and two mistranslation products in Bacillus thuringiensis BMB171. However, its function and promoter regulation is still not precise. In this study, we demonstrated that the encoding product of the rppA gene acts as an ARE1 ABC-F protein and confers resistance to antibiotics virginiamycin M1 and lincomycin when overexpressed. Besides the reported attenuator LRR, the expression of the rppA gene is controlled by the sigma factor SigA and a global transcription factor CcpA. Consequently, its promoter activity is mainly maintained at the stationary phase of cell growth and inhibited in the presence of glucose. Our study revealed the function and regulation of the rppA gene in detail. KEY POINTS: • The RppA protein acts as an ARE1 ABC-F protein • The rppA gene confers resistance to antibiotics virginiamycin M1 and lincomycin when overexpressed • The expression of the rppA gene is regulated by the sigma factor SigA and the pleiotropic regulator CcpA.

Role of Hfq in glucose utilization, biofilm formation and quorum sensing system in Bacillus subtilis.

Hfq is an RNA-binding protein, its main function is to participate in post-transcriptional regulation of bacteria and regulate small regulatory RNA (sRNA) and messenger RNA (mRNA) stability, but the Hfq function of Bacillus subtilis (B. subtilis) has not been fully explained. In this study, we used the strains of B. subtilis168 (BS168), BS168Δhfq and BS168Δhfq-C to explore the effects of Hfq on the glucose utilization, biofilm formation and quorum sensing (QS) system of B. subtilis. The results showed that the knockout of hfq resulted in growth defects when bacteria were cultured in the Luria-Bertani (LB) medium, but we did not observe the same effects in Nitrogen medium (NM) and Inorganic Salt-free medium (ISM). We further found that the growth of strains under different glucose concentrations was also different, which was related to the expression of CcpA. Interestingly, the hfq mutant showed increased resistance to a high-glucose environment. Furthermore, the biofilm and extracellular poly saccharides (EPS) formation of BS168Δhfq decreased significantly. At the same time, changes were observed in the morphology of the biofilm, such as larger intercellular space of the biofilm and thinner edge. The qRT-PCR results confirmed that the hfq knockout caused significant up-regulation or down-regulation of gene expression in QS system, and down-regulated genes were involved in the positive regulation of biofilm formation. Taken together, we demonstrated that Hfq plays a vital role in glucose utilization, biofilm formation and QS of B. subtilis, which provides a new perspective for subsequent related research.

Carbon Source-Dependent Reprogramming of Anaerobic Metabolism in Staphylococcus aureus.

To be a successful pathogen, Staphylococcus aureus has to adapt its metabolism to the typically oxygen- and glucose-limited environment of the host. Under fermenting conditions and in the presence of glucose, S. aureus uses glycolysis to generate ATP via substrate-level phosphorylation and mainly lactic acid fermentation to maintain the redox balance by reoxidation of NADH equivalents. However, it is less clear how S. aureus proceeds under anoxic conditions and glucose limitation, likely representing the bona fide situation in the host. Using a combination of proteomic, transcriptional, and metabolomic analyses, we show that in the absence of an abundant glycolysis substrate, the available carbon source pyruvate is converted to acetyl coenzyme A (AcCoA) in a pyruvate formate-lyase (PflB)-dependent reaction to produce ATP and acetate. This process critically depends on derepression of the catabolite control protein A (CcpA), leading to upregulation of pflB transcription. Under these conditions, ethanol production is repressed to prevent wasteful consumption of AcCoA. In addition, our global and quantitative characterization of the metabolic switch prioritizing acetate over lactate fermentation when glucose is absent illustrates examples of carbon source-dependent control of colonization and pathogenicity factors.IMPORTANCE Under infection conditions, S. aureus needs to ensure survival when energy production via oxidative phosphorylation is not possible, e.g., either due to the lack of terminal electron acceptors or by the inactivation of components of the respiratory chain. Under these conditions, S. aureus can switch to mixed-acid fermentation to sustain ATP production by substrate level phosphorylation. The drop in the cellular NAD+/NADH ratio is sensed by the repressor Rex, resulting in derepression of fermentation genes. Here, we show that expression of fermentation pathways is further controlled by CcpA in response to the availability of glucose to ensure optimal resource utilization under growth-limiting conditions. We provide evidence for carbon source-dependent control of colonization and virulence factors. These findings add another level to the regulatory network controlling mixed-acid fermentation in S. aureus and provide additional evidence for the lifestyle-modulating effect of carbon sources available to S. aureus.

Metabolism of Poly-ß1,4-N-Acetylglucosamine Substrates and Importation of N-Acetylglucosamine and Glucosamine by Enterococcus faecalis.

The ability of Enterococcus faecalis to use a variety of carbon sources enables colonization at various anatomic sites within a mammalian host. N-Acetylglucosamine (GlcNAc) is one of the most abundant natural sugars and provides bacteria with a source of carbon and nitrogen when metabolized. N-Acetylglucosamine is also a component of bacterial peptidoglycan, further highlighting the significance of N-acetylglucosamine utilization. In this study, we show that CcpA-regulated enzymes are required for growth on the poly-ß1,4-linked GlcNAc substrate, chitopentaose (ß1,4-linked GlcNAc5). We also show that EF0114 (EndoE) is required for growth on chitobiose (ß1,4-linked GlcNAc2) and that the GH20 domain of EndoE is required for the conversion of GlcNAc2 to N-acetylglucosamine. GlcNAc is transported into the cell via two separate phosphotransferase system (PTS) complexes, either the PTS IICBA encoded by ef1516 (nagE) or the Mpt glucose/mannose permease complex (MptBACD). The Mpt PTS is also the primary glucosamine transporter. In order for N-acetylglucosamine to be utilized as a carbon source, phosphorylated N-acetylglucosamine (GlcNAc-6-P) must be deacetylated, and here, we show that this activity is mediated by EF1317 (an N-acetylglucosamine-6-phosphate deacetylase; NagA homolog), as a deletion of ef1317 is unable to grow on GlcNAc as the carbon source. Deamination of glucosamine to fructose-6-phosphate is required for entry into glycolysis, and we show that growth on glucosamine is dependent on EF0466 (a glucosamine-6-phosphate deaminase; NagB homolog). Collectively, our data highlight the chitinolytic machinery required for breaking down exogenous chitinous substrates, as well as the uptake and cytosolic enzymes needed for metabolizing N-acetylglucosamine. IMPORTANCE Enterococcus faecalis causes life-threatening health care-associated infections in part due to its intrinsic and acquired antibiotic resistance, its ability to form biofilms, and its nutrient versatility. Alternative nutrient acquisition systems are key factors that contribute to enterococcal colonization at biologically unique host anatomic sites. Although E. faecalis can metabolize an array of carbon sources, little is known of how this bacterium acquires these secondary nutrient sources in mammalian hosts. Our research identifies the glycosidase machinery required for degrading exogenous chitinous substrates into N-acetylglucosamine monomers for transport and metabolism of one of the most abundant naturally occurring sugars, N-acetylglucosamine. Disrupting the function of this N-acetylglucosamine acquisition pathway may lead to new treatments against multidrug-resistant enterococcal infections.

Activity of CcpA-Regulated GH18 Family Glycosyl Hydrolases That Contributes to Nutrient Acquisition and Fitness in Enterococcus faecalis.

The ability of Enterococcus faecalis to colonize host anatomical sites is dependent on its adaptive response to host conditions. Three glycosyl hydrolase gene clusters, each belonging to glycosyl hydrolase family 18 (GH18) (ef0114, ef0361, and ef2863), in E. faecalis were previously found to be upregulated under glucose-limiting conditions. The GH18 catalytic domain is present in proteins that are classified as either chitinases or ß-1,4 endo-ß-N-acetylglucosaminidases (ENGases) based on their ß-1,4 endo-N-acetyl-ß-d-glucosaminidase activity, and ENGase activity is commonly associated with cleaving N-linked glycoprotein, an abundant glycan structure on host epithelial surfaces. Here, we show that all three hydrolases are negatively regulated by the transcriptional regulator carbon catabolite protein A (CcpA). Additionally, we demonstrate that a constitutively active CcpA variant represses the expression of CcpA-regulated genes irrespective of glucose availability. Previous studies showed that the GH18 catalytic domains of EndoE (EF0114) and EfEndo18A (EF2863) were capable of deglycosylating RNase B, a model high-mannose-type glycoprotein. However, it remained uncertain which glycosidase is primarily responsible for the deglycosylation of high-mannose-type glycoproteins. In this study, we show by mutation analysis as well as a dose-dependent analysis of recombinant protein expression that EfEndo18A is primarily responsible for deglycosylating high-mannose glycoproteins and that the glycans removed by EfEndo18A support growth under nutrient-limiting conditions in vitro. In contrast, IgG is representative of a complex-type glycoprotein, and we demonstrate that the GH18 domain of EndoE is primarily responsible for the removal of this glycan decoration. Finally, our data highlight the combined contribution of glycosidases to the virulence of E. faecalis in vivo.

Glutamate Dehydrogenase (GdhA) of Streptococcus pneumoniae Is Required for High Temperature Adaptation.

During its progression from the nasopharynx to other sterile and nonsterile niches of its human host, Streptococcus pneumoniae must cope with changes in temperature. We hypothesized that the temperature adaptation is an important facet of pneumococcal survival in the host. Here, we evaluated the effect of temperature on pneumococcus and studied the role of glutamate dehydrogenase (GdhA) in thermal adaptation associated with virulence and survival. Microarray analysis revealed a significant transcriptional response to changes in temperature, affecting the expression of 252 genes in total at 34°C and 40°C relative to at 37°C. One of the differentially regulated genes was gdhA, which is upregulated at 40°C and downregulated at 34°C relative to 37°C. Deletion of gdhA attenuated the growth, cell size, biofilm formation, pH survival, and biosynthesis of proteins associated with virulence in a temperature-dependent manner. Moreover, deletion of gdhA stimulated formate production irrespective of temperature fluctuation. Finally, ΔgdhA grown at 40°C was less virulent than other temperatures or the wild type at the same temperature in a Galleria mellonella infection model, suggesting that GdhA is required for pneumococcal virulence at elevated temperature.

Deciphering the Regulation of the Mannitol Operon Paves the Way for Efficient Production of Mannitol in Lactococcus lactis.

Lactococcus lactis has great potential for high-yield production of mannitol, which has not yet been fully realized. In this study, we characterize how the mannitol genes in L. lactis are organized and regulated and use this information to establish efficient mannitol production. Although the organization of the mannitol genes in L. lactis was similar to that in other Gram-positive bacteria, mtlF and mtlD, encoding the enzyme IIA component (EIIAmtl) of the mannitol phosphotransferase system (PTS) and the mannitol-1-phosphate dehydrogenase, respectively, were separated by a transcriptional terminator, and the mannitol genes were found to be organized in two transcriptional units: an operon comprising mtlA, encoding the enzyme IIBC component (EIIBCmtl) of the mannitol PTS, mtlR, encoding a transcriptional activator, and mtlF, as well as a separately expressed mtlD gene. The promoters driving expression of the two transcriptional units were somewhat similar, and both contained predicted catabolite responsive element (cre) genes. The presence of carbon catabolite repression was demonstrated and was shown to be relieved in stationary-phase cells. The transcriptional activator MtlR (mtlR), in some Gram-positive bacteria, is repressed by phosphorylation by EIIAmtl, and when we knocked out mtlF, we indeed observed enhanced expression from the two promoters, which indicated that this mechanism was in place. Finally, by overexpressing the mtlD gene and using stationary-phase cells as biocatalysts, we attained 10.1 g/liter mannitol with a 55% yield, which, to the best of our knowledge, is the highest titer ever reported for L. lactis. Summing up, the results of our study should be useful for improving the mannitol-producing capacity of this important industrial organism. IMPORTANCE Lactococcus lactis is the most studied species of the lactic acid bacteria, and it is widely used in various food fermentations. To date, there have been several attempts to persuade L. lactis to produce mannitol, a sugar alcohol with important therapeutic and food applications. Until now, to achieve mannitol production in L. lactis with significant titer and yield, it has been necessary to introduce and express foreign genes, which precludes the use of such strains in foods, due to their recombinant status. In this study, we systematically characterize how the mannitol genes in L. lactis are regulated and demonstrate how this impacts mannitol production capability. We harnessed this information and managed to establish efficient mannitol production without introducing foreign genes.

Regulation of CcpA on the growth and organic acid production characteristics of ruminal Streptococcus bovis at different pH.

BACKGROUND: Catabolite control protein A (CcpA) regulates the transcription of lactate dehydrogenase and pyruvate formate-lyase in Streptococcus bovis, but knowledge of its role in response to different pH is still limited. In this study, a ccpA-knockout strain of S. bovis S1 was constructed and then used to examine the effects of ccpA gene deletion on the growth and fermentation characteristics of S. bovis S1 at pH 5.5 or 6.5. RESULTS: There was a significant interaction between strain and pH for the maximum specific growth rate (µmax) and growth lag period (λ), which caused a lowest µmax and a longest λ in ccpA-knockout strain at pH 5.5. Deletion of ccpA decreased the concentration and molar percentage of lactic acid, while increased those of formic acid. Strains at pH 5.5 had decreased concentrations of lactic acid and formic acid compared to pH 6.5. The significant interaction between strain and pH caused the highest production of total organic acids and acetic acid in ccpA-knockout strain at pH 6.5. The activities of α-amylase and lactate dehydrogenase decreased in ccpA-knockout strain compared to the wild-type strain, and increased at pH 5.5 compared to pH 6.5. There was a significant interaction between strain and pH for the activity of acetate kinase, which was the highest in the ccpA-knockout strain at pH 6.5. The expression of pyruvate formate-lyase and acetate kinase was higher in the ccpA-knockout strain compared to wild-type strain. The lower pH improved the relative expression of pyruvate formate-lyase, while had no effect on the relative expression of acetate kinase. The strain × pH interaction was significant for the relative expression of lactate dehydrogenase and α-amylase, both of which were highest in the wild-type strain at pH 5.5 and lowest in the ccpA-knockout strain at pH 6.5. CONCLUSIONS: Overall, low pH inhibited the growth of S. bovis S1, but did not affect the fermentation pattern. CcpA regulated S. bovis S1 growth and organic acid fermentation pattern. Moreover, there seemed to be an interaction effect between pH and ccpA deletion on regulating the growth and organic acids production of S. bovis S1.

Lactic acid production using cheese whey based medium in a stirred tank reactor by a ccpA mutant of Lacticaseibacillus casei.

In lactobacilli, CcpA is known to modulate the expression of genes involved in sugar metabolism, stress response and aerobic adaptation. This study aimed to evaluate a ccpA mutant of Lacticaseibacillus casei BL23 to increase lactic acid production using cheese whey. The ccpA derivative (BL71) showed better growth than the L. casei wild-type in the whey medium. In a stirred tank reactor, at 48 h, lactate production by BL71 was eightfold higher than that by BL23. In batch fermentations, the final values reached were 44.23 g L-1 for BL71 and 27.58 g L-1 for BL23. Due to a decrease in the delay of lactate production in the mutant, lactate productivity increased from 0.17 g (L.h)-1 with BL23 to 0.80 g (L.h)-1 with BL71. We found that CcpA would play additional roles in nitrogen metabolism by the regulation of the proteolytic system. BL71 displayed higher activity of the PepX, PepQ and PrtP enzymes than BL23. Analysis of prtP expression confirmed this deregulation in BL71. Promoter analysis of the prtP gene revealed CcpA binding sites with high identity to the cre consensus sequence and the interaction of CcpA with this promoter was confirmed in vitro. We postulate that deregulation of the proteolytic system in BL71 allows a better exploitation of nitrogen resources in cheese whey, resulting in enhanced fermentation capacity. Therefore, the ccpA gene could be a good target for future technological developments aimed at effective and inexpensive lactate production from dairy industrial wastes.