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Peptidoglycan (PG) is an essential bacterial architecture pivotal for shape maintenance and adaptation to osmotic stress. Although PG synthesis and modification are tightly regulated under harsh environmental stresses, few related mechanisms have been investigated. In this study, we aimed to investigate the coordinated and distinct roles of the PG carboxypeptidases DacC and DacA, in adaptation to alkaline and salt stresses and shape maintenance in Escherichia coli. We found that DacC is an alkaline PG carboxypeptidase, whose enzyme activity and protein stability are significantly enhanced under alkaline stress. Both DacC and DacA were required for bacterial growth under alkaline stress, whereas only DacA was required for the adaptation to salt stress. Under normal growth conditions, only DacA was necessary for cell shape maintenance, while under alkaline stress conditions, both DacA and DacC were necessary for cell shape maintenance, but their roles were distinct. Notably, all these roles of DacC and DacA were independent of LD-transpeptidases, which are necessary for the formation of PG 3-3 crosslinks and covalent bonds between PG and the outer membrane lipoprotein Lpp. Instead, DacC and DacA interacted with penicillin-binding proteins (PBPs), DD-transpeptidases, mostly in a C-terminal domain-dependent manner, and these interactions were necessary for most of their roles. Collectively, our results demonstrate the coordinated and distinct novel roles of PG carboxypeptidases in stress adaptation and shape maintenance and provide novel insights into the cellular functions of PG carboxypeptidases associated with PBPs.

Vancomycin and β-lactams are clinically important antibiotics that inhibit the formation of peptidoglycan cross-links, but their binding targets are different. The binding target of vancomycin is D-alanine-D-alanine (D-Ala-D-Ala), whereas that of β-lactam is penicillin-binding proteins (PBPs). In this study, we revealed the divergent effects of peptidoglycan (PG) carboxypeptidases on vancomycin and β-lactam resistance in Escherichia coli and Bacillus subtilis. The deletion of PG carboxypeptidases induced sensitivity to most β-lactams, whereas it induced strong resistance toward vancomycin. Notably, both of two phenotypes did not have strong association with LD-transpeptidases, which are necessary for the formation of PG 3-3 cross-links and covalent bonds between PG and an Lpp outer membrane (OM) lipoprotein. Vancomycin resistance was induced by increased amount of decoy D-Ala-D-Ala residues within PG, whereas β-lactam sensitivity was associated with physical interactions between PG carboxypeptidase and PBPs. The presence of OM permeability barrier strongly strengthened vancomycin resistance, but it significantly weakened β-lactam sensitivity. Collectively, our results revealed two distinct LD-transpeptidase-independent functions of PG carboxypeptidases, which involved inverse modulation of bacterial resistance to clinically important antibiotics, β-lactams and vancomycin, and presented evidence for a link between PG carboxypeptidase and PBPs.IMPORTANCEBacterial peptidoglycan (PG) hydrolases play important roles in various aspects of bacterial physiology, including cytokinesis, PG synthesis, quality control of PG, PG recycling, and stress adaptation. Of all the PG hydrolases, the role of PG carboxypeptidases is poorly understood, especially regarding their impacts on antibiotic resistance. To date, most studies on PG carboxypeptidases are focused on LD-transpeptidase-related roles. We have revealed two distinct LD-transpeptidase-independent functions of PG carboxypeptidases with respect to antibiotic resistance. The deletion of PG carboxypeptidases led to sensitivity to most β-lactams, while it caused strong resistance to vancomycin. The underlying molecular mechanisms of two phenotypes were not associated with LD-transpeptidases. Therefore, our study provides novel insights into the roles of PG carboxypeptidases in the regulation of antibiotic resistance and a potential clue for the development of a drug to improve the clinical efficacy of β-lactam antibiotics.One sentence summaryEffect of peptidoglycan carboxypeptidase on antibiotic

Although bacteria have diverse membrane proteins, the function of many of them remains unknown or uncertain even in Escherichia coli. In this study, to investigate the function of hypothetical membrane proteins, genome-wide analysis of phenotypes of hypothetical membrane proteins was performed under various envelope stresses. Several genes responsible for adaptation to envelope stresses were identified. Among them, deletion of YhcB, a conserved inner membrane protein of unknown function, caused high sensitivities to various envelope stresses and increased membrane permeability, and caused growth defect under normal growth conditions. Furthermore, yhcB deletion resulted in morphological aberration, such as branched shape, and cell division defects, such as filamentous growth and the generation of chromosome-less cells. The analysis of antibiotic susceptibility showed that the yhcB mutant was highly susceptible to various anti-folate antibiotics. Notably, all phenotypes of the yhcB mutant were completely or significantly restored by YhcB without the transmembrane domain, indicating that the localization of YhcB on the inner membrane is dispensable for its function. Taken together, our results demonstrate that YhcB is involved in cell morphology and cell division in a membrane localization-independent manner.

Although many β-agarases that hydrolyze the β-1,4 linkages of agarose have been biochemically characterized, only three α-agarases that hydrolyze the α-1,3 linkages are reported to date. In this study, a new α-agarase, AgaWS5, from Catenovulum sediminis WS1-A, a new agar-degrading marine bacterium, was biochemically characterized. AgaWS5 belongs to the glycoside hydrolase (GH) 96 family. AgaWS5 consists of 1295 amino acids (140 kDa) and has the 65% identity to an α-agarase, AgaA33, obtained from an agar-degrading bacterium Thalassomonas agarivorans JAMB-A33. AgaWS5 showed the maximum activity at a pH and temperature of 8 and 40 °C, respectively. AgaWS5 showed a cold-tolerance, and it retained more than 40% of its maximum enzymatic activity at 10 °C. AgaWS5 is predicted to have several calcium-binding sites. Thus, its activity was slightly enhanced in the presence of Ca2+, and was strongly inhibited by EDTA. The Km and Vmax of AgaWS5 for agarose were 10.6 mg/mL and 714.3 U/mg, respectively. Agarose-liquefication, thin layer chromatography, and mass and NMR spectroscopic analyses demonstrated that AgaWS5 is an endo-type α-agarase that degrades agarose and mainly produces agarotetraose. Thus, in this study, a novel cold-adapted GH96 agarotetraose-producing α-agarase was identified.

The unified saccharification and fermentation (USF) system was developed for direct production of ethanol from agarose. This system contains an enzymatic saccharification process that uses three types of agarases and a fermentation process by recombinant yeast. The pGMFα-HGN plasmid harboring AGAH71 and AGAG1 genes encoding β-agarase and the NABH558 gene encoding neoagarobiose hydrolase was constructed and transformed into the Saccharomyces cerevisiae 2805 strain. Three secretory agarases were produced by introducing an S. cerevisiae signal sequence, and they efficiently degraded agarose to galactose, 3,6-anhydro- L-galactose (AHG), neoagarobiose, and neoagarohexose. To directly produce ethanol from agarose, the S. cerevisiae 2805/pGMFα-HGN strain was cultivated into YP-containing agarose medium at 40°C for 48 h (for saccharification) and then 30°C for 72 h (for fermentation). During the united cultivation process for 120 h, a maximum of 1.97 g/l ethanol from 10 g/l agarose was produced. This is the first report on a single process containing enzymatic saccharification and fermentation for direct production of ethanol without chemical liquefaction (pretreatment) of agarose.

Agar, a major polysaccharide of red algal cells, is degraded by β-agarases into neoagarobiose, which is further hydrolyzed into the monomers, d-galactose and 3,6-anhydro-l-galactose, by 1,3-α-3,6-anhydro-l-galactosidases including α-1,3-l-neoagarooligasaccharide hydrolase (α-NAOSH). A novel cold-adapted alkaline α-NAOSH, Ahg558, consisting of 359 amino acids (40.8 kDa) was identified from Gayadomonas joobiniege G7. It was annotated as a glycosyl hydrolase family 43 based on genomic sequence analysis, showing 84% and 74% identities with the characterized α-NAOSHs from Agarivorans gilvus WH0801 and Saccharophagus degradans 2–40, respectively. The recombinant Ahg558 (rAhg558) purified from Escherichia coli formed dimers and cleaved α-1,3 glycosidic bonds at the non-reducing ends of the neoagarobiose, neoagarotetraose, and neoagarohexaose, which was confirmed by thin-layer chromatography and mass spectrometry. The optimum pH and temperature for rAhg558 activity were 9.0 and 30 °C, respectively. Unusually, it retained over 93% activity in a broad range of temperatures between 0 and 40 °C and over 73% in a broad range of pH between pH 6.0 and pH 9.0, indicating it is a unique cold-adapted alkaline exo-acting α-NAOSH. Its enzymatic activity was dependent on Mn2+ ions. Km and Vmax values toward neoagarobiose were 2.6 mg/mL (8.01 mM) and 133.33 U/mg, respectively.

Phosphorylation is the most important modification for protein regulation; it controls many signal transduction pathways in all organisms. While several tools to detect phosphorylated proteins have been developed to study a variety of basic cellular processes involving protein phosphorylation, these methods have several limitations. Many proteins exhibit a phosphorylation-dependent electrophoretic mobility shift (PDEMS) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the molecular mechanism responsible for this phenomenon has been elucidated recently. The method for detecting phosphorylated proteins can be simplified by the application of the PDEMS. Herein, we present a novel simple method to detect protein phosphorylation, which is based on the construction of a variant protein displaying a PDEMS. The PDEMS of proteins is caused by the distribution of negatively charged amino acids around the phosphorylation site, i.e. an electrophoretic mobility shift (EMS)-related motif (ΘX1-3ΘX1-3Θ, where Θ corresponds to an acidic or phosphorylated amino acid and X represents any amino acid). The EMS-related motif can be constructed by the introduction of a negative charge by phosphorylation; it results in the decreased binding of SDS to the proteins, consequently inducing the retardation of the mobility of the protein during SDS-PAGE. Based on these molecular analyses of the PDEMS, a protein with the EMSrelated motif is designed and used to determine the in vivo phosphorylation state of the protein. This method may be used as a general strategy to easily measure the ratio of protein phosphorylation in cells.

Peptidoglycan (PG) hydrolases play important roles in various aspects of bacterial physiology, including cytokinesis, PG synthesis, quality control of PG, PG recycling, and antibiotic resistance. However, the regulatory mechanisms of their expression are poorly understood. In this study, we have uncovered novel regulatory mechanisms of the protein levels of the synthetically lethal PG endopeptidases MepS and MepM, which are involved in PG synthesis. A mutant defective for both MepS and MepM was lethal in an amino acid-rich medium, whereas it exhibited almost normal growth in a minimal medium, suggesting the expendability of MepS and MepM in a minimal medium. Protein levels of MepS and MepM dramatically decreased in the minimal medium. Although MepM was revealed as a substrate of Prc, a periplasmic protease involved in the proteolysis of MepS, only the decrease in the MepS level in the minimal medium was affected by theprcdepletion. Phenotypic and biochemical analyses showed that the presence of aromatic amino acids in the medium induced the accumulation of MepS, but not MepM, while the presence of glutamate increased the level of MepM, but not MepS. Together, these results demonstrate that the protein levels of the two major PG endopeptidases are regulated in an amino acid availability-dependent manner, but their molecular mechanisms and signaling are significantly distinct.

The inner membrane protein lipopolysaccharide assembly protein B (LapB) is an adaptor protein that activates the proteolysis of LpxC by an essential inner membrane metalloprotease, FtsH, leading to a decrease in the level of lipopolysaccharide in the membrane. In this study, we revealed the mechanism by which the essential inner membrane protein YejM regulates LapB and analyzed the role of the transmembrane domain of LapB in Escherichia coli. The transmembrane domain of YejM genetically and physically interacted with LapB and inhibited its function, which led to the accumulation of LpxC. The transmembrane domain of LapB was indispensable for both its physical interaction with YejM and its regulation of LpxC proteolysis. Notably, we found that the lapB mutant exhibited strong cold sensitivity and this phenotype was not associated with increased accumulation of LpxC. The transmembrane domain of LapB was also required for its role in adaptation to cold stress. Taken together, these results showed that LapB plays an important role in both the regulation of LpxC level, which is controlled by its interaction with the transmembrane domain of YejM, and adaptation to cold stress, which is independent of LpxC.

Actinobacteria utilize various polysaccharides in the soil as carbon source by degrading them via extracellular hydrolytic enzymes. Agarose, a marine algal polysaccharide composed of D-galactose and 3,6-anhydro-L-galactose (AHG), is one of the carbon sources used by S. coelicolor A3(2). However, little is known about agar hydrolysis in S. coelicolor A3(2), except that the regulation of agar hydrolysis metabolism is strongly inhibited by glucose as in the catabolic pathways of other polysaccharides. In this study, we elucidated the role of DagR in regulating the expression of three agarase genes (dagA, dagB, and dagC) in S. coelicolor A3(2) by developing a dagR-deletion mutant (Δsco3485). We observed that the Δsco3485 mutant had increased mRNA level of the agarolytic pathway genes and 1.3-folds higher agarase production than the wild type strain, indicating that the dagR gene encodes a cluster-suited repressor. Electrophoretic mobility shift assay revealed that DagR bound to the upstream regions of the three agarase genes. DNase 1 footprinting analysis demonstrated that a palindromic sequence present in the upstream region of the three agarase genes was essential for DagR-binding. Uniquely, the DNA-binding activity of DagR was inhibited by AHG, one of the final degradation products of agarose. AHG-induced agarase production was not observed in the Δsco3485 mutant, as opposed to that in the wild type strain. Therefore, DagR acts as a repressor that binds to the promoter region of the agarase genes, inhibits gene expression at the transcriptional level, and is derepressed by AHG. This is the first report on the regulation of gene expression regarding agar metabolism in S. coelicolor A3(2).

Agarose is a linear polysaccharide composed of D-galactose and 3,6-anhydro-L-galactose (AHG). It is a major component of the red algal cell wall and is gaining attention as an abundant marine biomass. However, the inability to ferment AHG is considered an obstacle in the large-scale use of agarose and could be addressed by understanding AHG catabolism in agarolytic microorganisms. Since AHG catabolism was uniquely confirmed in

1,3-α-3,6-anhydro-L-galactosidase (α-neoagarooligosaccharide hydrolase) catalyzes the last step of agar degradation by hydrolyzing neoagarobiose into monomers, D-galactose, and 3,6-anhydro-Lgalactose, which is important for the bioindustrial application of algal biomass. Ahg943, from the agarolytic marine bacterium

Gram-negative bacteria have an outer membrane inhibiting the entry of antibiotics. Porins, found within the outer membrane, are involved in regulating the permeability of &beta, lactam antibiotics. &beta, lactamases are enzymes that are able to inactivate the antibacterial properties of &beta, lactam antibiotics. Interestingly, porins and &beta, lactamase are found in outer membrane vesicles (OMVs) of &beta, lactam-resistant Escherichia coli and may be involved in the survival of susceptible strains of E. coli in the presence of antibiotics, through the hydrolysis of the &beta, lactam antibiotic. In this study, OMVs isolated from &beta, lactam-resistant E. coli and from mutants, lacking porin or &beta, lactamase, were evaluated to establish if the porins or &beta, lactamase in OMVs were involved in the degradation of &beta, lactam antibiotics. OMVs isolated from E. coli deficient in &beta, lactamase did not show any degradation ability against &beta, lactam antibiotics, while OMVs lacking OmpC or OmpF showed significantly lower levels of hydrolyzing activity than OMVs from parent E. coli. These data reveal an important role of OMVs in bacterial defense mechanisms demonstrating that the OmpC and OmpF proteins allow permeation of &beta, lactam antibiotics into the lumen of OMVs, and antibiotics that enter the OMVs can be degraded by &beta, lactamase.

A novel β-agarase, AgaJ5, was identified from an agar-degrading marine bacterium, Gayadomonas joobiniege G7. It belongs to the glycoside hydrolase family 86 and is composed of 805 amino acids with a 30-amino-acid signal peptide. Zymogram analysis showed that purified AgaJ5 has agarase activity. The optimum temperature and pH for AgaJ5 activity were determined to be 30°C and 4.5, respectively. AgaJ5 was an acidic β-agarase that had strong activity at a narrow pH range of 4.5-5.5, and was a cold-adapted enzyme, retaining 40% of enzymatic activity at 10°C. AgaJ5 required monovalent ions such as Na+ and K+ for its maximum activity, but its activity was severely inhibited by several metal ions. The Km and Vmax of AgaJ5 for agarose were 8.9 mg/ml and 188.6 U/mg, respectively. Notably, thin-layer chromatography, mass spectrometry, and agarose-liquefication analyses revealed that AgaJ5 was an endo-type β-agarase producing neoagarohexaose as the final main product of agarose hydrolysis. Therefore, these results suggest that AgaJ5 from G. joobiniege G7 is a novel endo-type neoagarohexaose-producing β-agarase having specific biochemical features that may be useful for industrial applications.

The sco6546 gene of Streptomyces coelicolor A3(2) was annotated as a putative glycosyl hydrolase belonging to family 48. It is predicted to encode a 973-amino acid polypeptide (103.4 kDa) with a 39-amino acid secretion signal. Here, the SCO6546 protein was overexpressed in Streptomyces lividans TK24, and the purified protein showed the expected molecular weight of the mature secreted form (934 aa, 99.4 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. SCO6546 showed high activity toward Avicel and carboxymethyl cellulose, but low activity toward filter paper and β-glucan. SCO6546 showed maximum cellulase activity toward Avicel at pH 5.0 and 50 °C, which is similar to the conditions for maximum activity toward cellotetraose and cellopentaose substrates. The kinetic parameters kcat and KM , for cellotetraose at pH 5.0 and 50 °C were 13.3 s-1 and 2.7 mM, respectively. Thin layer chromatography (TLC) of the Avicel hydrolyzed products generated by SCO6546 showed cellobiose only, which was confirmed by mass spectral analysis. TLC analysis of the cello-oligosaccharide and chromogenic substrate hydrolysates generated by SCO6546 revealed that it can hydrolyze cellodextrins mainly from the non-reducing end into cellobiose. These data clearly demonstrated that SCO6546 is an exo-β-1,4-cellobiohydrolase (EC 3.2.1.91), acting on nonreducing end of cellulose.

The microbial production of renewable ethylene glycol (EG) has been gaining attention recently due to its growing importance in chemical and polymer industries. EG has been successfully produced biosynthetically from d-xylose through several novel pathways. The first report on EG biosynthesis employed the Dahms pathway in Escherichia coli wherein 71% of the theoretical yield was achieved. This report further improved the EG yield by implementing metabolic engineering strategies. First, d-xylonic acid accumulation was reduced by employing a weak promoter which provided a tighter control over Xdh expression. Second, EG yield was further improved by expressing the YjgB, which was identified as the most suitable aldehyde reductase endogenous to E. coli. Finally, cellular growth, d-xylose consumption, and EG yield were further increased by blocking a competing reaction. The final strain (WTXB) was able to reach up to 98% of the theoretical yield (25% higher as compared to the first study), the highest reported value for EG production from d-xylose.

Agar, a major component of the cell wall of red algae, is an interesting heteropolysaccharide containing an unusual sugar, 3,6-anhydro-L-galactose. It is widely used as a valuable material in various industrial and experimental applications due to its characteristic gelling and stabilizing properties. Agar-derived oligosaccharides or mono-sugars produced by various agarases have become a promising subject for research owing to their unique biological activities, including anti-obesity, anti-diabetic, immunomodulatory, anti-tumor, antioxidant, skin-whitening, skin-moisturizing, anti-fatigue, and anti-cariogenic activities. Agar is also considered as an alternative sustainable source of biomass for chemical feedstock and biofuel production to substitute for the fossil resource. In this review, we summarize various biochemically characterized agarases, which are useful for industrial applications, such as neoagarooligosaccharide or agarooligosaccharide production and saccharification of agar. Additionally, we succinctly discuss various recent studies that have been conducted to investigate the versatile biological activities of agar-derived saccharides and biofuel production from agar biomass. This review provides a basic framework for understanding the importance of agarases and agar-derived saccharides with broad applications in pharmaceutical, cosmetic, food, and bioenergy industries.

How motile bacteria recognize their environment and decide whether to stay or navigate toward more favorable location is a fundamental issue in survival. The flagellum is an elaborate molecular device responsible for bacterial locomotion, and the flagellum-driven motility allows bacteria to move themselves to the appropriate location at the right time. Here, we identify the polar landmark protein HubP as a modulator of polar flagellation that recruits the flagellar assembly protein FapA to the old cell pole, thereby controlling its activity for the early events of flagellar assembly in Vibrio vulnificus. We show that dephosphorylated EIIAGlc of the PEP-dependent sugar transporting phosphotransferase system sequesters FapA from HubP in response to glucose and hence inhibits FapA-mediated flagellation. Thus, flagellar assembly and motility is governed by spatiotemporal control of FapA, which is orchestrated by the competition between dephosphorylated EIIAGlc and HubP, in the human pathogen V. vulnificus.

The sco0765 gene was annotated as a glycosyl hydrolase family 5 endoglucanase from the genomic sequence of Streptomyces coelicolor A3(2) and consisted of 2,241 bp encoding a polypeptide of 747 amino acids (molecular weight of 80.5 kDa) with a 29-amino acid signal peptide for secretion. The SCO0765 recombinant protein was heterogeneously over-expressed in Streptomyces lividans TK24 under the control of a strong ermE* promoter. The purified SCO0765 protein showed the expected molecular weight of the mature form (718 aa, 77.6 kDa) on sodium dodecyl sulfate-polyacryl amide gel electrophoresis. SCO0765 showed high activity toward β-glucan and carboxymethyl cellulose (CMC) and negligible activity to Avicel, xylan, and xyloglucan. The SCO0765 cellulase had a maximum activity at pH 6.0 and 40°C toward CMC and at pH 9.0 and 50-60°C toward β-glucan. Thin layer chromatography of the hydrolyzed products of CMC and β-glucan by SCO0765 gave cellotriose as the major product and cellotetraose, cellopentaose, and longer oligosaccharides as the minor products. These results clearly demonstrate that SCO0765 is an endo-β-1,4-cellulase, hydrolyzing the β-1,4 glycosidic bond of cellulose into cellotriose.

To survive in a continuously changing environment, bacteria sense concentration gradients of attractants or repellents, and purposefully migrate until a more favourable habitat is encountered. While glucose is known as the most effective attractant, the flagellar biosynthesis and hence chemotactic motility has been known to be repressed by glucose in some bacteria. To date, the only known regulatory mechanism of the repression of flagellar synthesis by glucose is via downregulation of the cAMP level, as shown in a few members of the family Enterobacteriaceae. Here we show that, in Vibrio vulnificus, the glucose-mediated inhibition of flagellar motility operates by a completely different mechanism. In the presence of glucose, EIIA(Glc) is dephosphorylated and inhibits the polar localization of FapA (flagellar assembly protein A) by sequestering it from the flagellated pole. A loss or delocalization of FapA results in a complete failure of the flagellar biosynthesis and motility. However, when glucose is depleted, EIIA(Glc) is phosphorylated and releases FapA such that free FapA can be localized back to the pole and trigger flagellation. Together, these data provide new insight into a bacterial strategy to reach and stay in the glucose-rich area.

Gayadomonas joobiniege G7 is an agar-degrading bacterium, which produces various agarases that have been biochemically characterized recently. In this study, we biochemically characterized a new β-agarase AgaJ10 belonging to the glycoside hydrolase (GH) 42 family from G. joobiniege G7. AgaJ10 is composed of 762 amino acids (89 kDa) and has the highest similarity (63% identity) to a putative β-agarase from the agar-degrading bacterium Catenovulum sp. DS-2, which was obtained from the intestines of a Haliotis diversicolor. The optimal pH and temperature for AgaJ10 activity were determined to be 5.0 and 30 °C, respectively. AgaJ10 exhibited a cold tolerance, retaining more than 40% of its enzymatic activity at 5 °C. The Km and Vmax of AgaJ10 for agarose were 61.5 mg/mL and 294.1 U/mg, respectively. Notably, the activity of AgaJ10 was significantly enhanced by Mn2+ but was strongly inhibited by some metal ions, including Fe2+, Ni2+, and Cu2+. Agarose-liquefaction, mass spectrometry, and thin-layer chromatography analyses showed that AgaJ10 is an exo-type β-agarase that hydrolyzes agarose only into neoagarobiose. Therefore, this study is the first report of a GH42 β-agarase that catalyzes a neoagarobiose-producing exo-type reaction.

Gram-negative pathogens, such as Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii, pose a serious threat to public health worldwide, due to high rates of antibiotic resistance and the lack of development of novel antimicrobial agents targeting Gram-negative bacteria. The outer membrane (OM) of Gram-negative bacteria is a unique architecture that acts as a potent permeability barrier against toxic molecules, such as antibiotics. The OM is composed of phospholipids, lipopolysaccharide (LPS), outer membrane β-barrel proteins (OMP), and lipoproteins. These components are synthesized in the cytoplasm or in the inner membrane, and are then selectively transported to the OM by the specific transport machines, including the Lol, BAM, and Lpt pathways. In this review, we summarize recent studies on the assembly systems of OM components and analyze studies for the development of inhibitors that target these systems. These analyses show that OM assembly machines have the potential to be a novel attractive drug target of Gram-negative bacteria.

Agar is a major polysaccharide of red algal cells and is mainly decomposed into neoagarobiose by the co-operative effort of β-agarases. Neoagarobiose is hydrolyzed into monomers, d-galactose and 3,6-anhydro-l-galactose, via a microbial oxidative process. Therefore, the enzyme, 1,3-α-3,6-anhydro-l-galactosidase (α-neoagarobiose/neoagarooligosaccharide hydrolase) involved in the final step of the agarolytic pathway is crucial for bioindustrial application of agar. A novel cold-adapted α-neoagarooligosaccharide hydrolase, Ahg786, was identified and characterized from an agarolytic marine bacterium Gayadomonas joobiniege G7. Ahg786 comprises 400 amino acid residues (45.3 kDa), including a 25 amino acid signal peptide. Although it was annotated as a hypothetical protein from the genomic sequencing analysis, NCBI BLAST search showed 57, 58, and 59% identities with the characterized α-neoagarooligosaccharide hydrolases from Saccharophagus degradans 2–40, Zobellia galactanivorans, and Bacteroides plebeius, respectively. The signal peptide-deleted recombinant Ahg786 expressed and purified from Escherichia coli showed dimeric forms and hydrolyzed neoagarobiose, neoagarotetraose, and neoagarohexaose into 3,6-anhydro-l-galactose and other compounds by cleaving α-1,3-glycosidic bonds from the non-reducing ends of neoagarooligosaccharides, as confirmed by thin-layer chromatography and mass spectrometry. The optimum pH and temperature for Ahg786 activity were 7.0 and 15 °C, respectively, indicative of its unique cold-adapted features. The enzymatic activity severely inhibited with 0.5 mM ethylenediaminetetraacetic acid was completely restored or remarkably enhanced by Mn2+ in a concentration-dependent manner, suggestive of the dependence of the enzyme on Mn2+ ions. Km and Vmax values for neoagarobiose were 4.5 mM and 1.33 U/mg, respectively.

The progressive increase of antibiotic resistance poses an alarming threat on public health worldwide. Although antibiotic resistance is a problem of human health, a one health approach to tackling antibiotic resistance is required, due to the effect of animal and environment on human health. To exactly estimate the impact of animal and environment on the spread of antibiotic resistance in human, scientific evidence with high quality is required. Molecular analysis of antibiotic-resistant bacteria through high-resolution, such as whole-genome sequencing, is suitable for obtaining credible evidence in examining the transmission of strains or genetic mobile elements responsible for antibiotic resistance among human, animal, and environment. In this paper, we discuss many observational studies using the whole-genome sequencing on various isolates collected from human, animal, and environment. These analyses suggest that more extensive surveillance based on whole-genome sequencing is required to accurately assess the potential threat of animal and environment on the spread of antibiotic resistance in human and that the effort to overcome various limitations in obtaining higher quality evidence is required.

Fast detection of β-lactamase ( bla ) genes allows improved surveillance studies and infection control measures, which can minimize the spread of antibiotic resistance. Although several molecular diagnostic methods have been developed to detect limited bla gene types, these methods have significant limitations, such as their failure to detect almost all clinically available bla genes. We developed a fast and accurate molecular method to overcome these limitations using 62 primer pairs, which were designed through elaborate optimization processes. To verify the ability of this large-scale bla detection method ( large-scale bla Finder), assays were performed on previously reported bacterial control isolates/strains. To confirm the applicability of the large-scale bla Finder, the assays were performed on unreported clinical isolates. With perfect specificity and sensitivity in 189 control isolates/strains and 403 clinical isolates, the large-scale bla Finder detected almost all clinically available bla genes. Notably, the large-scale bla Finder detected 24 additional unreported bla genes in the isolates/strains that were previously studied, suggesting that previous methods detecting only limited types of bla genes can miss unexpected bla genes existing in pathogenic bacteria, and our method has the ability to detect almost all bla genes existing in a clinical isolate. The ability of large-scale bla Finder to detect bla genes on a large scale enables prompt application to the detection of almost all bla genes present in bacterial pathogens. The widespread use of the large-scale bla Finder in the future will provide an important aid for monitoring the emergence and dissemination of bla genes and minimizing the spread of resistant bacteria.

Widespread antimicrobial use and concomitant resistance have led to a significant threat to public health. Because inappropriate use and overuse of antibiotics based on insufficient knowledge are one of the major drivers of antibiotic resistance, education about prudent antibiotic use aimed at both the prescribers and the public is important. This review investigates recent studies on the effect of interventions for promoting prudent antibiotics prescribing. Up to now, most educational efforts have been targeted to medical professionals, and many studies showed that these educational efforts are significantly effective in reducing antibiotic prescribing. Recently, the development of educational programs to reduce antibiotic use is expanding into other groups, such as the adult public and children. The investigation of the contents of educational programs for prescribers and the public demonstrates that it is important to develop effective educational programs suitable for each group. In particular, it seems now to be crucial to develop appropriate curricula for teaching medical and nonmedical (pharmacy, dentistry, nursing, veterinary medicine, and midwifery) undergraduate students about general medicine, microbial virulence, mechanism of antibiotic resistance, and judicious antibiotic prescribing.

A novel β-agarase, AgaJ5, was identified from an agar-degrading marine bacterium

Klebsiella pneumoniae is one of the most clinically relevant species in immunocompromised individuals responsible for community-acquired and nosocomial infections, including pneumonias, urinary tract infections, bacteremias, and liver abscesses. Since the mid-1980s, hypervirulent K. pneumoniae, generally associated with the hypermucoviscosity phenotype, has emerged as a clinically significant pathogen responsible for serious disseminated infections, such as pyogenic liver abscesses, osteomyelitis, and endophthalmitis, in a generally younger and healthier population. Hypervirulent K. pneumoniae infections were primarily found in East Asia and now are increasingly being reported worldwide. Although most hypervirulent K. pneumoniae isolates are antibiotic-susceptible, some isolates with combined virulence and resistance, such as the carbapenem-resistant hypervirulent K. pneumoniae isolates, are increasingly being detected. The combination of multidrug resistance and enhanced virulence has the potential to cause the next clinical crisis. To better understand the basic biology of hypervirulent K. pneumoniae, this review will provide a summarization and discussion focused on epidemiology, hypervirulence-associated factors, and antibiotic resistance mechanisms of such hypervirulent strains. Epidemiological analysis of recent clinical isolates in China warns the global dissemination of hypervirulent K. pneumoniae strains with extensive antibiotic resistance in the near future. Therefore, an immediate response to recognize the global dissemination of this hypervirulent strain with resistance determinants is an urgent priority.

Acinetobacter baumannii is undoubtedly one of the most successful pathogens responsible for hospital-acquired nosocomial infections in the modern healthcare system. Due to the prevalence of infections and outbreaks caused by multi-drug resistant A. baumannii, few antibiotics are effective for treating infections caused by this pathogen. To overcome this problem, knowledge of the pathogenesis and antibiotic resistance mechanisms of A. baumannii is important. In this review, we summarize current studies on the virulence factors that contribute to A. baumannii pathogenesis, including porins, capsular polysaccharides, lipopolysaccharides, phospholipases, outer membrane vesicles, metal acquisition systems, and protein secretion systems. Mechanisms of antibiotic resistance of this organism, including acquirement of -lactamases, up-regulation of multidrug efflux pumps, modification of aminoglycosides, permeability defects, and alteration of target sites, are also discussed. Lastly, novel prospective treatment options for infections caused by multi-drug resistant A. baumannii are summarized.

Preferential sugar utilization is a widespread phenomenon in biological systems. Glucose is usually the most preferred carbon source in various organisms, especially in bacteria where it is taken up via the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The currently proposed model for glucose preference over non-PTS sugars in enteric bacteria including E. coli is strictly dependent on the phosphorylation state of the glucose-specific PTS component, enzyme IIAGlc (EIIAGlc). However, the mechanism of the preference among PTS sugars is largely unknown in Gram-negative bacteria. Here, we show that glucose preference over another PTS sugar, mannitol, is absolutely dependent on the general PTS component HPr, but not on EIIAGlc, in E. coli. Dephosphorylated HPr accumulates during the transport of glucose and interacts with the mannitol operon regulator, MtlR, to augment its repressor activity. This interaction blocks the inductive effect of mannitol on the mannitol operon expression and results in the inhibition of mannitol utilization.

The bacterial enzyme RppH initiates mRNA decay by removing pyrophosphate from 5΄-triphosphorylated mRNA. Escherichia coli RppH has promiscuous substrate specificity, but relatively few transcripts are affected by loss of RppH. The phenotypic analysis of the rppH mutant is required for understanding the physiological role of RppH, but the phenotype of the rppH mutant has not yet been determined. In this study, we provide several phenotypes of the rppH mutant associated with envelope integrity. Through phenotype analysis and drug susceptibility testing, we found that the rppH mutant is sensitive to a variety of chemicals including antibiotics, and is also significantly sensitive to envelope stresses, such as osmotic stress, ethanol and sodium dodecyl sulfate. All phenotypes of the rppH mutant were caused by loss of its enzymatic activity. The rppH mutant exhibited increased envelope permeability, compared to wild-type cells. In contrast, an increase of RppH activity significantly inhibited the growth of wild-type cells under low-temperature conditions. In conclusion, various phenotypes of the rppH mutant propose that RppH is associated with regulation of envelope integrity.

The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) is a multicomponent system that participates in a variety of physiological processes in addition to the phosphorylation-coupled transport of numerous sugars. In Escherichia coli and other enteric bacteria, enzyme IIA Glc (EIIA Glc ) is known as the central processing unit of carbon metabolism and plays multiple roles, including regulation of adenylyl cyclase, the fermentation/respiration switch protein FrsA, glycerol kinase, and several non-PTS transporters, whereas the only known regulatory role of the E. coli histidine-containing phosphocarrier protein HPr is in the activation of glycogen phosphorylase. Because HPr is known to be more abundant than EIIA Glc in enteric bacteria, we assumed that there might be more regulatory mechanisms connected with HPr. The ligand fishing experiment in this study identified Rsd, an anti-sigma factor known to complex with σ 70 in stationary-phase cells, as an HPr-binding protein in E. coli . Only the dephosphorylated form of HPr formed a tight complex with Rsd and thereby inhibited complex formation between Rsd and σ 70 . Dephosphorylated HPr, but not phosphorylated HPr, antagonized the inhibitory effect of Rsd on σ 70 -dependent transcriptions both in vivo and in vitro, and also influenced the competition between σ 70 and σ S for core RNA polymerase in the presence of Rsd. Based on these data, we propose that the anti-σ 70 activity of Rsd is regulated by the phosphorylation state-dependent interaction of HPr with Rsd.

While many eukaryotic and some prokaryotic proteins show a phosphorylation-dependent mobility shift (PDMS) on SDS-PAGE, the molecular mechanism for this phenomenon had not been elucidated. We have recently shown that the distribution of negatively charged amino acids around the phosphorylation site is important for the PDMS of some proteins. Here, we show that replacement of the phosphorylation site with a negatively charged amino acid results in a similar degree of the mobility shift of a protein as phosphorylation, indicating that the PDMS is due to the introduction of a negative charge by phosphorylation. Compared with a protein showing no shift, one showing a retarded mobility on SDS-PAGE had a decreased capacity for SDS binding. The elucidation of the consensus sequence (ΘX1-3ΘX1-3Θ, where Θ corresponds to an acidic function) for a PDMS suggests a general strategy for mutagenizing a phosphorylatable protein resulting in a PDMS.

Summary In addition to the phosphoenolpyruvate:sugar phosphotransferase system (sugar PTS), most proteobacteria possess a paralogous system (nitrogen phosphotransferase system, PTSNtr). The first proteins in both pathways are enzymes (enzyme Isugar and enzyme INtr) that can be autophosphorylated by phosphoenolpyruvate. The most striking difference between enzyme Isugar and enzyme INtr is the presence of a GAF domain at the N-terminus of enzyme INtr. Since the PTSNtr was identified in 1995, it has been implicated in a variety of cellular processes in many proteobacteria and many of these regulations have been shown to be dependent on the phosphorylation state of PTSNtr components. However, there has been little evidence that any component of this so-called PTSNtr is directly involved in nitrogen metabolism. Moreover, a signal regulating the phosphorylation state of the PTSNtr had not been uncovered. Here, we demonstrate that glutamine and α-ketoglutarate, the canonical signals of nitrogen availability, reciprocally regulate the phosphorylation state of the PTSNtr by direct effects on enzyme INtr autophosphorylation and the GAF signal transduction domain is necessary for the regulation of enzyme INtr activity by the two signal molecules. Taken together, our results suggest that the PTSNtr senses nitrogen availability.

Fast detection of β-lactamase (bla) genes allows improved surveillance studies and infection control measures, which can minimize the spread of antibiotic resistance. Although several molecular diagnostic methods have been developed to detect limited bla gene types, these methods have significant limitations, such as their failure to detect almost all clinically available bla genes. We developed a fast and accurate molecular method to overcome these limitations using 62 primer pairs, which were designed through elaborate optimization processes. To verify the ability of this large-scale bla detection method (large-scaleblaFinder), assays were performed on previously reported bacterial control isolates/strains. To confirm the applicability of the large-scaleblaFinder, the assays were performed on unreported clinical isolates. With perfect specificity and sensitivity in 189 control isolates/strains and 403 clinical isolates, the large-scaleblaFinder detected almost all clinically available bla genes. Notably, the large-scaleblaFinder detected 24 additional unreported bla genes in the isolates/strains that were previously studied, suggesting that previous methods detecting only limited types of bla genes can miss unexpected bla genes existing in pathogenic bacteria, and our method has the ability to detect almost all bla genes existing in a clinical isolate. The ability of large-scaleblaFinder to detect bla genes on a large scale enables prompt application to the detection of almost all bla genes present in bacterial pathogens. The widespread use of the large-scaleblaFinder in the future will provide an important aid for monitoring the emergence and dissemination of bla genes and minimizing the spread of resistant bacteria.

The nitrogen-metabolic phosphotransferase system, PTSNtr, consists of the enzymes INtr, NPr and IIANtr that are encoded by ptsP, ptsO, and ptsN, respectively. Due to the proximity of ptsO and ptsN to rpoN, the PTSNtr system has been postulated to be closely related with nitrogen metabolism. To define the correlation between PTSNtr and nitrogen metabolism, we performed ligand fishing with EIIANtr as a bait and revealed that D-glucosamine-6-phosphate synthase (GlmS) directly interacted with EIIANtr. GlmS, which converts D-fructose-6-phosphate (Fru6P) into D-glucosamine-6-phosphate (GlcN6P), is a key enzyme producing amino sugars through glutamine hydrolysis. Amino sugar is an essential structural building block for bacterial peptidoglycan and LPS. We further verified that EIIANtr inhibited GlmS activity by direct interaction in a phosphorylation-state-dependent manner. EIIANtr was dephosphorylated in response to excessive nitrogen sources and was rapidly degraded by Lon protease upon amino sugar depletion. The regulation of GlmS activity by EIIANtr and the modulation of glmS translation by RapZ suggest that the genes comprising the rpoN operon play a key role in maintaining amino sugar homeostasis in response to nitrogen availability and the amino sugar concentration in the bacterial cytoplasm.