A synthetic 5,3-cross-link in the cell wall of rod-shaped Gram-positive bacteria.

Gram-positive bacteria assemble a multilayered cell wall that provides tensile strength to the cell. The cell wall is composed of glycan strands cross-linked by nonribosomally synthesized peptide stems. Herein, we modify the peptide stems of the Gram-positive bacterium Bacillus subtilis with noncanonical electrophilic d-amino acids, which when in proximity to adjacent stem peptides form novel covalent 5,3-cross-links. Approximately 20% of canonical cell-wall cross-links can be replaced with synthetic cross-links. While a low level of synthetic cross-link formation does not affect B. subtilis growth and phenotype, at higher levels cell growth is perturbed and bacteria elongate. A comparison of the accumulation of synthetic cross-links over time in Gram-negative and Gram-positive bacteria highlights key differences between them. The ability to perturb cell-wall architecture with synthetic building blocks provides a novel approach to studying the adaptability, elasticity, and porosity of bacterial cell walls.

Clinical significance of coryneform Gram-positive rods from blood identified by MALDI-TOF mass spectrometry and their susceptibility profiles - a retrospective chart review.

With the advent of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), most Gram-positive rods (GPRs) are readily identified; however, their clinical relevance in blood cultures remains unclear. Herein, we assessed the clinical significance of GPRs isolated from blood and identified in the era of MALDI-TOF MS. A retrospective chart review of patients presenting to the Mayo Clinic, Rochester, MN, from January 1, 2013, to October 13, 2015, was performed. Any episode of a positive blood culture for a GPR was included. We assessed the number of bottles positive for a given isolate, time to positivity of blood cultures, patient age, medical history, interpretation of culture results by the healthcare team and whether infectious diseases consultation was obtained. We also evaluated the susceptibility profiles of a larger collection of GPRs tested in the clinical microbiology laboratory of the Mayo Clinic, Rochester, MN from January 1, 2013, to October 31, 2015. There were a total of 246 GPRs isolated from the blood of 181 patients during the study period. 56% (n = 101) were deemed contaminants by the healthcare team and were not treated; 33% (n = 59) were clinically determined to represent true bacteremia and were treated; and 8% (n = 14) were considered of uncertain significance, with patients prescribed treatment regardless. Patient characteristics associated with an isolate being treated on univariate analysis included younger age (P = 0.02), identification to the species level (P = 0.02), higher number of positive blood culture sets (P < 0.0001), lower time to positivity (P < 0.0001), immunosuppression (P = 0.03), and recommendation made by an infectious disease consultant (P = 0.0005). On multivariable analysis, infectious diseases consultation (P = 0.03), higher number of positive blood culture sets (P = 0.0005) and lower time to positivity (P = 0.03) were associated with an isolate being treated. 100, 83, 48 and 34% of GPRs were susceptible to vancomycin, meropenem, penicillin and ceftriaxone, respectively.

Bruker biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry system for identification of Nocardia, Rhodococcus, Kocuria, Gordonia, Tsukamurella, and Listeria species.

We evaluated whether the Bruker Biotyper matrix-associated laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system provides accurate species-level identifications of 147 isolates of aerobically growing Gram-positive rods (GPRs). The bacterial isolates included Nocardia (n = 74), Listeria (n = 39), Kocuria (n = 15), Rhodococcus (n = 10), Gordonia (n = 7), and Tsukamurella (n = 2) species, which had all been identified by conventional methods, molecular methods, or both. In total, 89.7% of Listeria monocytogenes, 80% of Rhodococcus species, 26.7% of Kocuria species, and 14.9% of Nocardia species (n = 11, all N. nova and N. otitidiscaviarum) were correctly identified to the species level (score values, ≥ 2.0). A clustering analysis of spectra generated by the Bruker Biotyper identified six clusters of Nocardia species, i.e., cluster 1 (N. cyriacigeorgica), cluster 2 (N. brasiliensis), cluster 3 (N. farcinica), cluster 4 (N. puris), cluster 5 (N. asiatica), and cluster 6 (N. beijingensis), based on the six peaks generated by ClinProTools with the genetic algorithm, i.e., m/z 2,774.477 (cluster 1), m/z 5,389.792 (cluster 2), m/z 6,505.720 (cluster 3), m/z 5,428.795 (cluster 4), m/z 6,525.326 (cluster 5), and m/z 16,085.216 (cluster 6). Two clusters of L. monocytogenes spectra were also found according to the five peaks, i.e., m/z 5,594.85, m/z 6,184.39, and m/z 11,187.31, for cluster 1 (serotype 1/2a) and m/z 5,601.21 and m/z 11,199.33 for cluster 2 (serotypes 1/2b and 4b). The Bruker Biotyper system was unable to accurately identify Nocardia (except for N. nova and N. otitidiscaviarum), Tsukamurella, or Gordonia species. Continuous expansion of the MALDI-TOF MS databases to include more GPRs is necessary.

Evaluation of the Bruker MALDI Biotyper for identification of Gram-positive rods: development of a diagnostic algorithm for the clinical laboratory.

Reported matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identification rates of Gram-positive rods (GPR) are low compared to identification rates of Gram-positive cocci. In this study, three sample preparation methods were compared for MALDI-TOF MS identification of 190 well-characterized GPR strains: direct transfer, direct transfer-formic acid preparation, and ethanol-formic acid extraction. Using the interpretation criteria recommended by the manufacturer, identification rates were significantly higher for direct transfer-formic acid preparation and ethanol-formic acid extraction than for direct transfer. Reducing the species cutoff from 2.0 to 1.7 significantly increased species identification rates. In a subsequent prospective study, 215 clinical GPR isolates were analyzed by MALDI-TOF MS, and the results were compared to those for identification using conventional methods, with discrepancies being resolved by 16S rRNA and rpoB gene analysis. Using the direct transfer-formic acid preparation and a species cutoff of 1.7, congruencies on the genus and species levels of 87.4% and 79.1%, respectively, were achieved. In addition, the rate of nonidentified isolates dropped from 12.1% to 5.6% when using an extended database, i.e., the Bruker database amended by reference spectra of the 190 GPR of the retrospective study. Our data demonstrate three ways to improve GPR identification by the Bruker MALDI Biotyper, (i) optimize sample preparation using formic acid, (ii) reduce cutoff scores for species identification, and (iii) expand the database. Based on our results, we suggest an identification algorithm for the clinical laboratory combining MALDI-TOF MS with nucleic acid sequencing.

Bhargavaea cecembensis gen. nov., sp. nov., isolated from the Chagos-Laccadive ridge system in the Indian Ocean.

A novel Gram-positive, rod-shaped, non-motile, non-spore-forming bacterium, strain DSE10(T), was isolated from a deep-sea sediment sample collected at a depth of 5904 m from the Chagos-Laccadive ridge system in the Indian Ocean. Cells of strain DSE10(T) were positive for catalase, oxidase, urease and lipase activities and contained iso-C(14 : 0), iso-C(15 : 0), iso-C(16 : 0) and anteiso-C(15 : 0) as the major fatty acids. The major respiratory quinones were MK-6 and MK-8 and the major lipids were phosphatidylglycerol and diphosphatidylglycerol. The cell-wall peptidoglycan contained diaminopimelic acid as the diagnostic diamino acid. A blast sequence similarity search based on 16S rRNA gene sequences indicated that the genera Planococcus, Planomicrobium, Bacillus and Geobacillus were the nearest phylogenetic neighbours to the novel isolate with gene sequence similarities ranging from 94.9 to 95.2 %. Phylogenetic analyses using neighbour-joining, minimum-evolution and maximum-parsimony methods indicated that strain DSE10(T) formed a deeply rooted lineage distinct from the clades represented by the genera Planococcus, Planomicrobium, Bacillus and Geobacillus. Further, strain DSE10(T) could be distinguished from the above-mentioned genera based on the presence of signature nucleotides G, A, C, T, C, A, G, C and T at positions 182, 444, 480, 492, 563, 931, 1253, 1300 and 1391, respectively, in the 16S rRNA gene sequence. Based on the phenotypic and phylogenetic characteristics determined in this study, strain DSE10(T) was assigned as the type species of a new genus, Bhargavaea gen. nov., as Bhargavaea cecembensis sp. nov. The type strain of Bhargavaea cecembensis gen. nov., sp. nov. is DSE10(T) (=LMG 24411(T)=JCM 14375(T)). The genomic DNA G+C content of strain DSE10(T) is 59.5+/-2.5 mol%.

Allofustis seminis gen. nov., sp. nov., a novel Gram-positive, catalase-negative, rod-shaped bacterium from pig semen.

An unknown Gram-positive, catalase-negative, facultatively anaerobic, non-spore-forming, rod-shaped bacterium originating from semen of a pig was characterized using phenotypic, molecular chemical and molecular phylogenetic methods. Chemical studies revealed the presence of a directly cross-linked cell wall murein based on L-lysine and a DNA G + C content of 39 mol%. Comparative 16S rRNA gene sequencing showed that the unidentified rod-shaped organism formed a hitherto unknown subline related, albeit loosely, to Alkalibacterium olivapovliticus, Alloiococcus otitis, Dolosigranulum pigrum and related organisms, in the low-G + C-content Gram-positive bacteria. However, sequence divergence values of > 11% from these recognized taxa clearly indicated that the novel bacterium represents a separate genus. Based on phenotypic and phylogenetic considerations, it is proposed that the unknown bacterium from pig semen be classified as a new genus and species, Allofustis seminis gen. nov., sp. nov. The type strain is strain 01-570-1(T) (= CCUG 45438(T) = CIP 107425(T)).

[Physiology of organotrophic and lithotrophic growth of the thermophilic iron-reducing bacteria Thermoterrabacterium ferrireducens and Thermoanaerobacter siderophilus]. / Fiziologiia organotrofnogo i litotrofnogo rosta termofil'nykh zhelezovosstanavlivaiushchikh bakterii Thermoterrabacterium ferrireducens i Thermoanaerobacter siderophilus.

Growth physiology of the iron-reducing bacteria Thermoterrabacterium ferrireducens and Thermoanaerobacter siderophilus was investigated. The stimulation of the organotrophic growth of T. ferrireducens and T. siderophilus in the presence of Fe(III) was shown to be due to the utilization of ferric iron as an electron acceptor in catabolic processes and not to the effect exerted on the metabolism by Fe(II) or by changes in the redox potential. It was established that Fe(III) reduction in T. ferrireducens is not a detoxication strategy. In T. siderophilus, this process is carried out to relieve the inihibitory effect of hydrogen. T. ferrireducens was shown to be capable of lithoautotrophic growth with molecular hydrogen as electron donor and amorphous ferric oxide as electron acceptor, in the absence of any organic substances. The minimum threshold of H2 consumption was 3 x 10(-5) vol % of H2. The presence of CO dehydrogenase activity in T. ferrireducens suggests that CO2 fixation in this organism involves the anaerobic acetyl-CoA pathway. T. siderophilus failed to grow under lithoautotrophic conditions. The fact that T. ferrireducens contains c-type cytochromes and T. sidrophilus lacks them confirms the operation of different mechanisms of ferric iron reduction in these species.

Why are rod-shaped bacteria rod shaped?

Generally speaking, bacteria grow and divide indefinitely, and as long as the growth conditions are maintained they retain constant dimensions and shapes with little variation. How they do this is a question that I have been considering for three decades. Here, I discuss two hypothetical mechanisms, one for Gram-positive rods and the other for Gram-negative rods. These mechanisms are consistent with what is known, but make some unproven assumptions.

Weissella soli sp. nov., a lactic acid bacterium isolated from soil.

Phylogenetic analysis of the 16S rRNA gene of bacterial isolates from garden soil showed relatedness to Weissella kandleri and Weissella confusa. However, the sequences had notable differences, and DNA-DNA hybridizations confirmed that the isolates are separate from these two species. The isolates could be further distinguished from all previously described Weissella species by electrophoretic analysis of whole-cell proteins, as well as by the results from different biochemical tests. The name Weissella soli is proposed for the new species, the type strain being Mi268T (= LMG 20113T = DSM 14420T).

Mechanism of anaerobic ether cleavage: conversion of 2-phenoxyethanol to phenol and acetaldehyde by Acetobacterium sp.

2-Phenoxyethanol is converted into phenol and acetate by a strictly anaerobic Gram-positive bacterium, Acetobacterium strain LuPhet1. Acetate results from oxidation of acetaldehyde that is the early product of the biodegradation process (Frings, J., and Schink, B. (1994) Arch. Microbiol. 162, 199-204). Feeding experiments with resting cell suspensions and 2-phenoxyethanol bearing two deuterium atoms at either carbon of the glycolic moiety as substrate demonstrated that the carbonyl group of the acetate derives from the alcoholic function and the methyl group derives from the adjacent carbon. A concomitant migration of a deuterium atom from C-1 to C-2 was observed. These findings were confirmed by NMR analysis of the acetate obtained by fermentation of 2-phenoxy-[2-(13)C,1-(2)H(2)]ethanol, 2-phenoxy-[1-(13)C,1-(2)H(2)]ethanol, and 2-phenoxy-[1,2-(13)C(2),1-(2)H(2)]ethanol. During the course of the biotransformation process, the molecular integrity of the glycolic unit was completely retained, no loss of the migrating deuterium occurred by exchange with the medium, and the 1,2-deuterium shift was intramolecular. A diol dehydratase-like mechanism could explain the enzymatic cleavage of the ether bond of 2-phenoxyethanol, provided that an intramolecular H/OC(6)H(5) exchange is assumed, giving rise to the hemiacetal precursor of acetaldehyde. However, an alternative mechanism is proposed that is supported by the well recognized propensity of alpha-hydroxyradical and of its conjugate base (ketyl anion) to eliminate a beta-positioned leaving group.

Structure of the Na+-driven flagellum from the homoacetogenic bacterium Acetobacterium woodii.

The Na+-dependent flagellum of Acetobacterium woodii was characterised. Flagellin and whole flagella were purified and analysed by SDS-PAGE and electron microscopy. The structure and dimensions of the filament and the hook-basal body, as revealed by electron microscopy, resemble those of H+-dependent flagella from gram-positive bacteria. Intramembrane particle rings were present at the cell pole in freeze-fractured A. woodii cells, which might correspond to the mot complex.

Purification and characterization of dipeptidyl aminopeptidase from Aureobacterium sp. WO26.

We isolated a bacterial strain with an enzyme which releases dipeptide from Gly-Arg-p-nitroanilide. The bacterium was tentatively identified as Aureobacterium sp. The enzyme, named AuDAP, was purified and characterized. It was homogenous by SDS-PAGE and IEF, and had a molecular mass of 90,000 Da by SDS-PAGE and 88,000 Da by gel filtration, so it may be a monomer. The isoelectric point was 3.8 and the optimum pH was 10.0. The purified enzyme hydrolyzed Gly-Arg-pNA, a model substrate for DAP I, and Arg-Arg-MNA, a model substrate for DAP III. However, this enzyme did not hydrolyze Gly-Phe pNA, also a model substrate for DAP I. These results suggested that this enzyme did not fall under the classification of mammalian DAPs and was similar to DAP BI from Pseudomonas sp. WO24 and dDAP from Dictyostelium discoideum, although several differences were observed between them. The N-terminal amino acid sequence of this enzyme showed no significant homology to any enzyme and protein, except only for DAP BI.

The phylogeny of Methanopyrus kandleri.

The phylogenetic position of Methanopyrus kandleri has been difficult to determine because reconstructions of phylogenetic trees based on rRNA sequences have been ambiguous. The most probable trees determined by most algorithms place the genus Methanopyrus at the base of a group that includes the halobacteria and the methanogens and their relatives, although occasionally some algorithms place this genus near the eocytes (the hyperthermophilic, sulfur-metabolizing prokaryotes), suggesting that it may belong to this lineage. In order to resolve the phylogeny of the genus Methanopyrus, we determined the sequence of an informative region of elongation factor 1-alpha that contains an 11-amino-acid insertion in eocytes and eukaryotes which is replaced by a 4-amino-acid insertion in methanogens, halobacteria, and eubacteria. On the basis of the results of our elongation factor 1-alpha gene analysis, we concluded that the genus Methanopyrus diverged from the eocyte branch before the eukaryotic and eocyte lineages separated and therefore is not an eocyte.

Biochemical characteristics and fatty acid composition of Gilardi rod group 1 bacteria.

Fifteen strains of eugonic, nonoxidative, gram-negative rods isolated primarily from human wounds of the extremities and blood formed a distinct group which was designated Gilardi rod group 1. The phenotypic characteristics of Gilardi rod group 1 were most similar to those of CDC group M-5, with the major difference that nitrite reduction was observed with CDC group M-5. All 15 strains of Gilardi rod group 1 possessed a distinct fatty acid profile which was characterized by large amounts (> 15%) of cis-vaccenic (18:1 omega 7c), palmitic (16:0), myristic (14:0), and lactobacillic (19:0 cyc11,12) acids and moderate amounts (3 to 5%) of lauric (12:0), 3-hydroxylauric (3-OH-12:0), and palmitoleic (16:1 omega 7c) acids. This fatty acid profile is unique compared with the profiles of CDC group M-5 and other bacteria we have tested and is useful for the rapid identification of Gilardi rod group 1 isolates.