The DNA sequence of the structural gene of gonococcal protein III and the flanking region containing a repetitive sequence. Homology of protein III with enterobacterial OmpA proteins.

The insert of a lambda gt11 clone expressing gonococcal protein III was sequenced. The deduced amino acid sequence showed a coding frame of 236 amino acids with a typical 22-amino-acid signal peptide, followed by the known NH2-terminal sequence of PIII. The mature protein has a molecular weight of 23,298. It was found that PIII had extensive and very striking homology to the carboxy-terminal portion of enterobacterial OmpA proteins. The homology encompasses the OmpA domain that is believed to be located in the periplasmic space. If the disposition of PIII across the OM is analogous, then the surface-exposed domain consists of less than 40 amino acids. These include a potential 15-amino-acid disulfide loop, a feature not found in OmpA proteins. Hybridization studies with the sequenced insert indicated that it contained a repetitive sequence that occurred at least 20 times in the genome. By additional hybridization studies the area containing the repetitive sequence was narrowed to a region of 43 bp. This region contained an exact copy of the consensus sequence of a 26-bp repetitive sequence recently described. An analogous sequence recurs in an inverted orientation 53 bp downstream.

Pyrolysis mass spectrometry of complex organic materials.

Pyrolysis mass spectrometry in combination with computerized multivariate statistical analysis enables qualitative and quantitative analysis of nonvolatile organic materials containing molecular assemblies of a complexity and size far beyond the capabilities of direct mass spectrometry. The state of the art in pyrolysis mass spectrometry techniques is illustrated through specific applications, including structural determination and quality control of synthetic polymers, quantitative analysis of polymer mixtures, classification and structural characterization of fossil organic matter, and nonsupervised numerical extraction of component patterns from complex biological samples.

Analysis of bacterial biotin-proteins.

The biotin-protein populations in several bacterial strains were analyzed by solubilization of [3H]biotin-labeled cells with sodium dodecylsulfate followed by electrophoresis on polyacrylamide gels containing the detergent. A variety of patterns of biotin-labeled polypeptide chains was seen, ranging from a single biotin-protein in Escherichia coli, corresponding to the biotin carboxyl carrier protein component of acetyl-CoA carboxylase, to multiple species in Enterobacter aerogenes, Pseudomonas citronellolis, Bacillus cereus, Propionibacterium shermanii, Lactobacillus plantarum, and Mycobacterium phlei, which probably represent subunits of multiple biotin-dependent enzymes present in these organisms. In the case of Pseudomonas citronellolis two major biotin-containing polypeptides with approximate molecular weights of 65 000 and 25 000 were shown to correspond to the biotin carboxyl carrier components of pyruvate carboxylase and acetyl-CoA carboxylase, respectively. Thus in the case of Pseudomonas citronellolis two different biotin-dependent enzymes in the same cell do not share common biotin carboxyl carrier subunits.

A conserved domain on enterobacterial porin protein analysed by monoclonal antibody.

Broadly cross-reactive monoclonal antibodies (MAbs) against enterobacterial outer membrane (OM) porin (Po) protein were isolated after immunization of BALB/c mice with whole cells of E. coli 055:B5. MAbs (n = 6) of the IgG class but of four different isotypes were studied. Based on a competition ELISA, all of the MAbs were directed against one and the same Po protein domain (Po I). The MAbs cross-reacted with 72 of 74 strains from 10 different genera of the Enterobacteriaceae. One Morganella and one Salmonella strain showed no cross-reactivity. Also, nine strains of various Neisseria spp. cross-reacted while 21 strains of various other nonenteric Gram-negative bacteria showed no cross-reactivity. The Po I sites were inaccessible in intact homologous bacteria but partially accessible in the OM. Digestion of OM with lysozyme or lysostaphin affected the accessibility of the Po I sites in OMs of various enterobacteria. Lysostaphin strongly enhanced the immunoaccessibility, whereas lysozyme had lesser effects. The enzymes also affected the binding by Neisseria OMs of the anti-Po I MAb. The Po I site was immunogenic both in humans and rabbits. The data indicate that Po I is an important Po protein domain, and that the effects of peptidoglycan-degrading enzymes must be considered in studies of Po protein domains.

Direct comparison of two mechanized systems for identification of gram-negative bacilli. Autobac ID system versus the auto microbic system (with EBC plus).

The Auto Microbic System (AMS) is an almost completely automated system, capable of identifying Enterobacteriaceae after 8 hours, and some glucose nonfermenters after 13 hours of incubation. The Autobac ID system is a mechanized, computer-assisted system capable of identifying Enterobacteriaceae and many nonfermentative gram-negative bacilli within 3-6 hours. The present report combines the results of two independent studies, both of which evaluated the AMS and the Autobac ID system compared with standard reference tests. Among the 1,510 isolates that were tested, both systems reported equivocal identifications (low confidence values) with 5-6% of the strains. AMS produced fewer erroneous identifications (3.8% vs. 4.9%) but more equivocal test results (5.7% vs. 4.9%). Reproducibility of the two systems was compared by triplicate testing of 88 selected strains in both laboratories. AMS was somewhat more reproducible than the Autobac ID system. The AMS was capable of identifying more species with greater accuracy and reproducibility, but the Autobac ID system was more rapid. Both systems demonstrated excellent accuracy and reproducibility and both could be used efficiently in the clinical laboratory.

A novel peptidoglycan-associated lipoprotein (PAL) found in the outer membrane of Proteus mirabilis and other Gram-negative bacteria.

A novel peptidoglycan-associated lipoprotein (PAL) was found in the cell envelope of Proteus mirabilis. This protein showed the following properties: (1) The apparent molecular weight in sodium dodecyl sulfate (SDS) polyacrylamide gel was 18,000. (2) The protein was present in the cell envelope in a form very closely, but not covalently, associated with the peptidoglycan layer. (3) The protein was recovered predominantly from the outer membrane fraction after separation of the cell envelopes. (4) [1-14C]Palmitic acid and [2-3H]glycerol were incorporated into the protein. (5) The protein contained covalently linked fatty acids (about 3 mol of fatty acid per mol of protein). (6) An unidentified compound was present in the hydrolysate of the protein. These properties, except for molecular weight and non-covalent association with the peptidoglycan, showed resemblance to those of Braun's lipoprotein. However the protein was distinct from Braun's lipoprotein in regard to amino acid composition. A similar peptidoglycan-associated lipoprotein (PAL) was present widely in the cell envelopes of various Gram-negative bacteria. P. mirabilis contains about twelve times as much PAL as Escherichia coli. Antiserum against PAL of P. mirabilis was cross-reactive against PAL of E. coli, but not against Braun's lipoprotein of E. coli.

Location and identification of substituents with free amino group in lipopolysaccharides of gram-negative bacteria with the use of chromophore labelling.

Interaction of ten different lipopolysaccharides (LPS) with 2,4-dinitrofluorobenzene yielded quantitatively yellow dinitrophenyl derivatives (DNP-LPS) to show the presence of substituents with free amino group. The DNP-LPS samples were degraded with 1% acetic acid, and after removal of lipid A precipitates the supernatants were separated on a Sephadex G-25 column to give coloured polysaccharide, oligosaccharide and monomeric fractions monitored at lambda DNP = 365 nm. The coloured materials, including DNP-derivative of lipid A, were dephosphorylated with hydrofluoric acid followed by identification of the released DNP-amines by thin layer chromatography (TLC) on silica gel. Subsequently, the dephosphorylated materials were hydrolysed with hydrochloric acid followed by TLC analysis. The approach allowed to detect, locate and identify the substituents with free amino group within the LPS molecules. Moreover, two types of core structures within LPS preparation from one strain were discovered for five microorganisms.

Protein profile typing--a new method of typing Morganella morganii strains.

A new, simple and stable method for typing Morganella morganii strains is described. The 150 strains examined, principally from faeces, contained haemolytic and non-haemolytic representatives of diverse O serogroup, bacteriocin type and biotype. Among the biotypes were some trehalose-fermenting, tetracycline-resistant strains and some non-motile, tetracycline-sensitive, glycerol fermenters. After analysis of cell lysates by sodium dodecyl sulphate-discontinuous polyacrylamide gel electrophoresis, strains could be differentiated into 21 types on the basis of outer membrane proteins (OMP) of 35-40 Kda. The OMP profile was not altered by culture on various common media and was unrelated to either O antigen or morganocin p-type. The finest strain recognition in M. morganii can be achieved by application of all three distinct typing methods.

Rahnella aquatilis, a potential contaminant in lager beer breweries.

The species Rahnella aquatilis has been isolated mostly from water, soil, and, in a few cases, from human clinical specimens; little is known about its ecological role. The application of polyacrylamide gel electrophoresis of soluble proteins, DNA-DNA hybridizations and API 20 E systems has shown that Rahnella aquatilis might also be encountered as a contaminant in lager beer breweries.

Structure of the O-specific polysaccharide from Enterobacter cloacae strain N.C.T.C. 11579 (serogroup O10).

The O-specific polysaccharide from the reference strain (N.C.T.C. 11579) for Enterobacter cloacae serogroup O10 has been isolated and characterised. By means of n.m.r. spectroscopy and methylation analysis, and by studies of the products obtained by Smith degradation or by N-deacetylation-deamination, the repeating unit of the polysaccharide could be allocated the structure shown. The polysaccharides from two cross-reacting serogroups (O9 and O11) have the same monosaccharide composition. (Formula: see text)

Outer membrane protein profiles of Edwardsiella ictaluri from fish.

Outer membrane proteins (OMP) prepared with sodium N-lauroyl sarcocinate (SLS) from 33 Edwardsiella ictaluri isolates from fish were examined by electrophoresis. Twenty-eight isolates from channel catfish (Ictalurus punctatus) had similar OMP profiles. Ten bands (71 kilodaltons [kD] to 19.5 kD) were identified in all isolates from channel catfish. One major 35-kD protein comprised most of the protein content of the outer membrane of isolates from channel catfish. Differences existed among isolates in the amount of protein within minor OMP bands. Edwardsiella ictaluri ATCC 33202 contained larger quantities of the 38.5- and 37-kD proteins than did the other isolates. Outer membrane protein profiles of E ictaluri derived from Bengal danio (Danio devario) and walking catfish (Clarias batrachus) were identical to OMP profiles of isolates from channel catfish. In contrast, OMP profiles from single isolates from green knife fish (Eigemannia virescens) and white catfish (Ictalurus catus) were different. Variations in incubation time, SLS extraction time, SLS extraction number, and in vivo and in vitro passage had no effect on the OMP profile of E ictaluri ATCC 33202. An increase in duration of sample solubilization did affect the OMP profile of E ictaluri ATCC 33202 by decreasing the amount of protein in 52-, 46-, and 43.5-kD bands. Accompanying the decrease were increased staining intensity in the 31.5- and 28.5-kD bands and the appearance of 4 new bands (34, 33, 25.5, and 22.5 kD). Edwardsiella ictaluri, a gram-negative bacterium in the family Enterobacteriaceae, is the cause of enteric septicemia of catfish.(ABSTRACT TRUNCATED AT 250 WORDS)

[Firefly assay of bacterial ATP: comparative study of extraction methods (author's transl)]. / Mesure de l'ATP bactérien par bioluminescence: étude critique des méthodes d'extraction.

The firefly bioluminescence ATP assay presents multiple applications in microbiological analysis. Nine ATP extraction methods were tested on thirteen Gram positive and Gram negative bacterial species. The DMSO method appears as the procedure for choice. It provides the highest yields and the best reproducibility, even at high bacterial concentrations. It is simple, rapid and can be recommended for routine analysis. Assays carried out after extraction by this procedure show that in 24 hours cultures, the ATP content ranges from 0.28 fg to 15.65 fg per cell depending on species.

[Analysis of cellular fatty acids of Enterobacteria species by gas chromatography-mass spectrometry].

Cellular fatty acid compositions of 15 Enterobacteria were analyzed by gas chromatography-mass spectrometry(GC-MS). About 30 fatty acids were detected in chromatograms, and 13 of them were chemically identified, e.i. C11:0, C12:0, C13:0, C14:0, C15:0, 2OH-C14:0, 3OH-C14:0, C16:1, C16:0, aC17:0, delta C17:0, C18:1 and C18:0. The major cellular fatty acids in all fifteen species were C16:0, C18:1, C15:0, C14:0, C16:1, C13:0, 3OH-C14:0 and C18:0. The cellular fatty acids of Enterobacteria species were characterized by normal straight-chain saturated acids and monounsaturated acids, of which the most abundant fatty acid was C16:0. 3OH-C14:0 was found in all strains of Enterobacteria, whereas 2OH-C14:0 was only found in strains of Serratia species. Other unknown compositions also would have been certain characteristics for bacteria. The present paper would provide some of useful reference data for chemotaxonomy and molecular microbiology of Enterobacteria.

[Polyamines in species of microorganisms of the intestinal group]. / Poliaminy nekotorykh vidov mikroorganizmov kishechnoi gruppy

A study was made of polyamines (putrescine, spermidine and spermin) in various representatives of the Enterobacteriaceae family--E. coli, Sh, sonnei, Sh. flexneri and S. typhi abdominalis. All the strains under study contained putrescine and spermidine, and many Shigella and Salmonella strains had spermin in addition. There were significant differences in the quantitative content of polyamines in the individual species. By the ratio of nitrogen of polyamine to phosphorus of nucleic acid it is possible to assess the correlation in the content of polyamines and nucleic acids in the bacterial cells.

[Heterogeneity of the fatty acid composition of bacteria in the genera Klebsiella, Enterobacter, Hafnia and Serratia]. / Geterogennost' zhirnokislotnogo sostava bakterii rodov Klebsiella, Enterobacter, Hafnia i Serratia.

The analysis of the chromatograms of methyl esters of fatty acids in bacterial strains of the tribe Klebsielleae showed the heterogeneity of the fatty acid composition of bacteria belonging to the genera Klebsiella, Enterobacter, Hafnia and Serratia. The bacteria of this tribe could be subdivided into 5 provisory groups in accordance with the composition and profile of their fatty acids. Group I comprised K. pneumoniae K1, K. ozaenae and K. rhinoscleromatis strains forming a separate group characterized by the absence of cyclopropane fatty acids; group II comprised K. pneumoniae strains of other capsular serovars (K2, K8, K11, K13, K41, K47), as well as K. aerogenes and K. oxytoca strains. The fatty acid composition of the strains of group III, comprising E. aerogenes and E. cloacae, was similar to that of E. coli O1; only among the fatty acids of these bacteria acid C20:0 could be detected. H. alvei strains were included into group IV; their fatty acid profiles were similar to those of the genus Enterobacter, but had a higher content of acids C15:0 and C18:0. S. marcescens strains were similar to the strains of group II in their fatty acid composition, but considerably differed from the latter by a higher content of hexadecanoate (C16:0) and a lower level of C18:1; for this reason they were regarded as provisory group V.

[Determination of the structural integrity of DNA from pathogenic enterobacteria]. / Opredelenie strukturnoi tselostnosti DNK patogennykh énterobakterii.

Among the enterobacterial strains under study, more organisms in the stationary phase of growth have been found to have nicks in their DNA than those in the exponential phase. Bacteria less sensitive to ultraviolet irradiation have the least number of nicks in each phase of growth. The number of nicks in different strains belonging to the serovar is sufficiently stable. Virulent and avirulent forms show no difference in this characteristic.