Bacterial lipoproteins are proteins that are post-translationally modified with a diacylglyceride at an N-terminal cysteine, which serves to tether these proteins to the outer face of the plasma membrane or to the outer membrane. This paper reviews recent insights into the enzymology of bacterial lipoprotein biosynthesis and localization. Moreover, we use bioinformatic analyses of bacterial lipoprotein signal peptide features and of the key biosynthetic enzymes to consider the distribution of lipoprotein biosynthesis at the phylum level. These analyses support the important conclusion that lipoprotein biosynthesis is a fundamental pathway utilized across the domain bacteria. Moreover, with the exception of a small number of sequences likely to derive from endosymbiont genomes, the enzymes of bacterial lipoprotein biosynthesis appear unique to bacteria, making this pathway an attractive target for the development of novel antimicrobials. Whilst lipoproteins with comparable signal peptide features are encoded in the genomes of Archaea, it is clear that these lipoproteins have a distinctive biosynthetic pathway that has yet to be characterized.
Bacteria ; classification ; enzymology ; metabolism ; Bacterial Proteins ; biosynthesis ; metabolism ; Enzymes ; metabolism ; Genome, Bacterial ; Lipoproteins ; biosynthesis ; metabolism ; Protein Sorting Signals

OBJECTIVETo compare the protein difference between B. henselae Houston and B. henselae Marseille by two-dimensional gel electrophoresis.

METHODProtein samples were prepared by vorterx, ultrasonic treatment, and centrifugation. Protein concentrations were determined by Bradford method. Protein difference was compared by the first IEF and the second SDS-polyacrylamide gel electrophoresis.

RESULTSProtein concentrations in samples of Bartonella henselae Houston and Bartonella henselae Marseille were 2.117 microg/microL and 2.200 microg/microL respectively. Sample protein of 40 microg for IPG strips loading was perfect. The results of 2-DE in pH 4 to 7 IPG strips showed that the total protein spots of Bartonella henselae Houston and Bartonella henselae Marseille were 375 and 379 respectively, 95% of the spots were the same between the two strains of Bartonella henselae.

CONCLUSIONThe procedure of 2-DE may prove successful for the proteomic analysis of Bartonella henselae. Bartonella henselae Houston and Bartonella henselae Marseille are different genotypes.

Bacterial Proteins ; analysis ; metabolism ; Bartonella henselae ; classification ; genetics ; metabolism ; Electrophoresis, Gel, Two-Dimensional ; Genotype ; Proteomics

OBJECTIVEYersinia enterocolitica is an extracellular pathogen and its related antigens interact with the host immune system. We investigated the difference in immunological characteristics between a highly pathogenic and poorly pathogenic strain of Y. enterocolitica.

METHODSWe used SDS-PAGE and western blotting to characterize lipopolysaccharide (LPS), Yersinia outer membrane proteins (Yops), membrane proteins, and whole-cell proteins from poorly pathogenic Y. enterocolitica bio-serotype 2/O:9, isolated from China, and highly pathogenic bio-serotype 1B/O:8, isolated from Japan.

RESULTSThese two strains of Y. enterocolitica had different LPS immune response patterns. Comparison of their Yops also showed differences that could have accounted for their differences in pathogenicity. The membrane and whole-cell proteins of both strains were similar; immunoblottting showed that the 35 kD and perhaps the 10 kD proteins were immunogens in both strains.

CONCLUSIONThe major antigens of the two strains eliciting the host immune response were the LPS and membrane proteins, as shown by comparing protein samples with reference and purified preparations.

Animals ; Antigens, Bacterial ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Blotting, Western ; Electrophoresis, Polyacrylamide Gel ; Female ; Gene Expression Regulation, Bacterial ; physiology ; Lipopolysaccharides ; metabolism ; Rabbits ; Yersinia enterocolitica ; classification ; metabolism

Magnetotactic bacteria (MTB), a group of phylogenetically diverse organisms that use their unique intracellular magnetosome organelles to swim along the Earth's magnetic field, play important roles in the biogeochemical cycles of iron and sulfur. Previous studies have revealed that the bacterial actin protein MamK plays essential roles in the linear arrangement of magnetosomes in MTB cells belonging to the Proteobacteria phylum. However, the molecular mechanisms of multiple-magnetosome-chain arrangements in MTB remain largely unknown. Here, we report that the MamK filaments from the uncultivated 'Candidatus Magnetobacterium casensis' (Mcas) within the phylum Nitrospirae polymerized in the presence of ATP alone and were stable without obvious ATP hydrolysis-mediated disassembly. MamK in Mcas can convert NTP to NDP and NDP to NMP, showing the highest preference to ATP. Unlike its Magnetospirillum counterparts, which form a single magnetosome chain, or other bacterial actins such as MreB and ParM, the polymerized MamK from Mcas is independent of metal ions and nucleotides except for ATP, and is assembled into well-ordered filamentous bundles consisted of multiple filaments. Our results suggest a dynamically stable assembly of MamK from the uncultivated Nitrospirae MTB that synthesizes multiple magnetosome chains per cell. These findings further improve the current knowledge of biomineralization and organelle biogenesis in prokaryotic systems.
Actins ; chemistry ; metabolism ; Adenosine Triphosphate ; metabolism ; Bacteria ; classification ; metabolism ; Bacterial Proteins ; chemistry ; metabolism ; Magnetospirillum ; classification ; metabolism ; Nucleotides ; metabolism ; Phylogeny ; Substrate Specificity

Pathogenic bacteria have increasingly been resisting to antimicrobial therapy. Recently, resistance problem has been relatively much worsened in Gram-negative bacilli. Acinetobacter spp. are typical nosocomial pathogens causing infections and high mortality, almost exclusively in compromised hospital patients. Acinetobacter spp. are intrinsically less susceptible to antibiotics than Enterobacteriaceae, and have propensity to acquire resistance. A surveillance study in Korea in 2009 showed that resistance rates of Acinetobacter spp. were very high: to fluoroquinolone 67%, to amikacin 48%, to ceftazidime 66% and to imipenem 51%. Carbapenem resistance was mostly due to OXA type carbapenemase production in A. baumannii isolates, whereas it was due to metallo-beta-lactamase production in non-baumannii Acinetobacter isolates. Colistin-resistant isolates were rare but started to be isolated in Korea. Currently, the infection caused by multidrug-resistant A. baumannii is among the most difficult ones to treat. Analysis at tertiary care hospital in 2010 showed that among the 1,085 isolates of Acinetobacter spp., 14.9% and 41.8% were resistant to seven, and to all eight antimicrobial agents tested, respectively. It is known to be difficult to prevent Acinetobacter spp. infection in hospitalized patients, because the organisms are ubiquitous in hospital environment. Efforts to control resistant bacteria in Korea by hospitals, relevant scientific societies and government agencies have only partially been successful. We need concerted multidisciplinary efforts to preserve the efficacy of currently available antimicrobial agents, by following the principles of antimicrobial stewardship.
Acinetobacter/classification/*drug effects/genetics/metabolism ; Anti-Bacterial Agents/pharmacology ; Bacterial Proteins/metabolism ; Drug Resistance, Multiple, Bacterial/genetics ; beta-Lactamases/metabolism

Cyclodextrin glucanotransferase, the essential enzyme for the production of cyclodextrins, has become the focus of scientific research nowadays. Although many related enzyme properties are well known, the crucial factors in product specificity determination remain to be answered. Here, the recent research progresses of cyclodextrin glucanotransferase, especially those about the evolution of product specificity, were reviewed, and the scientific problems were discussed.
Archaeal Proteins ; genetics ; metabolism ; Bacillus ; enzymology ; genetics ; Bacterial Proteins ; genetics ; metabolism ; Biocatalysis ; Cyclodextrins ; metabolism ; Evolution, Molecular ; Glucosyltransferases ; classification ; genetics ; metabolism ; Mutation ; Thermoanaerobacterium ; enzymology ; genetics ; Thermococcus

Microbial xylanases have received a great deal of attention in the last two decades for their potential applications in food, paper making and animal feed industries. Bacillus pumilus WL-11 was identified as a producer of alkane xylanase free of cellulase after screening soil samples of paper-making factories. The xylanase A (XylA) was purified to homogeneity from the culture filtrate of Bacillus pumilus WL-11 by (NH4) 2SO4 precipitation, CM-Sephadex and Sephadex G-75 chromatographies. The molecular mass of XylA is estimated to be 26.0 kD by SDS-PAGE and its isoelectric point is 9.5. The apparent Km is 16.6 mg/mL and V(max) is 1263 micromol/(min x mg) towards oat spelt xylan. XylA is optimally active between pH 7.2 and 8.0, and stable at pH 6.0 to 10.4. The enzyme is optimally active at 45 degrees C - 55 degrees C and stable at temperature below 45 degrees C, with its half time of activity of 35 min and 15 min at 55 degrees C and 60 degrees C respectively. HPLC analysis revealed that hydrolysis patterns of xylans from oat spelt, birch wood and beech wood by purified XylA were different. The XylA is determined to be an endo-beta-1,4-xylanase, as it generated mainly xylotriose and no xylose was detected among the three hydrolysates. XylA has strong hydrolytic activity towards the pentose in the hydrolysates of beech wood and birch wood xylans, but was not active to the pentose in the hydrolysate of oat spelt xylan. The crude WL-11 enzyme can efficiently hydrolyze oat spelt xylan to a series of xylo-oligosaccharides, suggesting its potential application in nutraceutical industry.
Bacillus ; classification ; enzymology ; Bacterial Proteins ; metabolism ; Culture Media ; Endo-1,4-beta Xylanases ; isolation & purification ; metabolism ; Substrate Specificity

OBJECTIVETo investigate the protein expression profiles of the major food-borne pathogen Campylobacter jejuni NCTC11168.

METHODSMembrane and soluble cellular proteins were extracted from the genome-sequenced C. jejuni strain NCTC11168. Protein expression profiles were determined using two-dimensional gel electrophoresis (2-DE). All the detected spots on the 2-DE map were subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF) analysis.

RESULTSA total of 537 and 333 spots were detected from the whole cell and membrane-associated proteins of C. jejuni NCTC11168 cultured on Columbia agar medium at 42 °C by 2-DE and Coomassie Brilliant Blue staining, respectively. Analyses of whole cell and membrane-associated proteins included 399 and 133 spots, respectively, which included 182 and 53 functional proteins identified by MALDI-TOF/TOF analysis.

CONCLUSIONThe comprehensive expression protein profiles of C. jeuni NCTC11168 obtained in this study will be useful for elucidating the roles of these proteins in further pathogenesis investigation.

Bacterial Proteins ; genetics ; metabolism ; Campylobacter jejuni ; classification ; genetics ; metabolism ; Electrophoresis, Gel, Two-Dimensional ; methods ; Gene Expression Regulation, Bacterial ; physiology ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; methods ; Transcriptome

OBJECTIVETo determine the frequency of mce gene in Leptospira interrogans, and to investigate the gene transcription levels of L. interrogans before and after infecting cells.

METHODSThe segments of entire mce genes from 13 L.interrogans strains and 1 L.biflexa strain were amplified by PCR and then sequenced after T-A cloning. A prokaryotic expression system of mce gene was constructed; the expression and output of the target recombinant protein rMce were examined by SDS-PAGE and Western Blot assay. Rabbits were intradermally immunized with rMce to prepare the antiserum, the titer of antiserum was measured by immunodiffusion test. The transcription levels of mce gene in L.interrogans serogroup Icterohaemorrhagiae serovar lai strain 56601 before and after infecting J774A.1 cells were monitored by real-time fluorescence quantitative RT-PCR.

RESULTmce gene was carried in all tested L.interrogans strains, but not in L.biflexa serogroup Semaranga serovar patoc strain Patoc I. The similarities of nucleotide and putative amino acid sequences of the cloned mce genes to the reported sequences (GenBank accession No: NP712236) were 99.02%-100% and 97.91%-100%, respectively. The constructed prokaryotic expression system of mce gene expressed rMce and the output of rMce was about 5% of the total bacterial proteins. The antiserum against whole cell of L.interrogans strain 56601 efficiently recognized rMce. After infecting J774A.1 cells, transcription levels of the mce gene in L.interrogans strain 56601 were remarkably up-regulated.

CONCLUSIONThe constructed prokaryotic expression system of mce gene and the prepared antiserum against rMce provide useful tools for further study of the gene function.

Amino Acid Sequence ; Animals ; Bacterial Proteins ; genetics ; metabolism ; DNA, Bacterial ; analysis ; Escherichia coli ; genetics ; metabolism ; Genes, Bacterial ; Leptospira interrogans ; classification ; genetics ; Molecular Sequence Data ; Rabbits ; Recombinant Proteins ; genetics ; metabolism ; Sequence Analysis, Protein ; Serotyping

OBJECTIVETo optimize the performance of Pulsed-Field Gel Electrophoresis (PFGE) for the comparison of inter-laboratory results and information exchange of Borrelia burgdorferi subtyping.

METHODSA panel of 34 strains of B. burgdorferi were used to optimize PFGE for subtyping. In order to optimize the electrophoretic parameters (EPs), all 34 strains of B. burgdorferi were analyzed using four EPs, yielding different Simpson diversity index (D) values and the epidemiological concordance was also evaluated.

RESULTSThe EP of a switch time of 1 s to 25 s for 13 h and 1 s to 10 s for 6 h produced the highest D value and was declared to be optimal for MluI and SmaI PFGE of B. burgdorferi. MluI and SmaI were selected as the first and second restriction enzymes for PFGE subtyping of B. burgdorferi according to discrimination and consistency with epidemiological data.

CONCLUSIONPFGE can be used as a valuable test for routine genospecies identification of B. burgdorferi.

Animals ; Bacterial Proteins ; metabolism ; Bacterial Typing Techniques ; Borrelia burgdorferi ; classification ; genetics ; isolation & purification ; DNA, Bacterial ; metabolism ; Deoxyribonucleases, Type II Site-Specific ; metabolism ; Electrophoresis, Gel, Pulsed-Field ; Humans ; Ixodes ; Rats