- Volume 133, Issue 5, 1987

The effects of low concentrations of O2 on fermentation in the cattle parasite Tritrichomonas foetus KV1 and its variant 1MR-100 were compared using membrane inlet mass spectrometry to measure simultaneously and continuously ethanol, CO2 and H2. In strain KV1 glucose-supported H2 and CO2 production were stimulated by O2 concentrations < 1·4 μm but were inhibited at higher concentrations. Damped oscillatory responses in H2 production indicated the operation of a feedback control system. Measurement of the O2-dependence of O2 consumption rates confirmed the presence of a high-affinity terminal oxidase (apparent K m = 1·6 μm-O2 at 37 °C) and substrate inhibition by O2 at > 8 μm-O2. Successive periods of exposure to O2 resulted in decreased O2 scavenging capacity, as indicated by increasing apparent K m values for O2. The variant strain 1MR-100 which lacks pyruvate: ferredoxin oxidoreductase and hydrogenase showed quite different characteristics: H2 production was not detectable, ethanol formation was inhibited by O2 (K i = 1 μm) and O2-dependence of O2 consumption indicated that no high-affinity oxidase was present (apparent K m = 33 μm-O2). Progressive increases in respiration rates on repeated exposure to low O2 concentrations indicated a capacity for adaptation to aerobiosis.

The arabinogalactan and peptidoglycan of armadillo-grown Mycobacterium leprae were examined. Within the limits defined by the small amount of material available, the resemblance of these polymers to those of other mycobacteria was confirmed. The polymers were linked by a highly acid-labile bond and the arabinogalactan was itself acid-labile; free arabinose and a variety of oligosaccharides containing both arabinose and galactose, as well as polysaccharide and peptidoglycan, were released by dilute acid. The resonances from anomeric protons in the proton NMR spectrum of the arabinogalactan were similar to those from the arabinogalactan of M. tuberculosis. The composition and structure of the peptidoglycan resembled those of other mycobacteria. The only major difference was the specific replacement of l-alanine by glycine in the peptide of the peptidoglycan.

The role of fructose 2,6-bisphosphate in the regulation of glycolysis in the citric acid accumulating fungus Aspergillus niger was investigated. Fructose 2,6-bisphosphate stimulated the activity of partially purified phosphofructokinase by increasing the affinity of the enzyme for fructose 6-phosphate and relieving inhibition by ATP. Fructose 2,6-bisphosphate acted synergistically with AMP, but not with NH+ 4 ions, which otherwise also activate phosphofructokinase. Fructose 2,6-bisphosphate also partially antagonized citrate inhibition of phosphofructokinase; complete deinhibition against high (5 mm) concentrations of citrate (as occur during citric acid accumulation), however, required the simultaneous presence of fructose 2,6-bisphosphate (0·1 μm). AMP (0·1 μm) and NH+ 4 ions (20 mm).

Porins are pore-forming outer-membrane proteins which serve as a non-specific pathway for the entry of hydrophilic molecules into Gram-negative bacteria. We studied four strains of Haemophilus influenzae that had decreased permeability to chloramphenicol associated with diminished quantities of a 40 kDa major outer-membrane protein. Isogenic pairs of organisms containing and lacking this protein were compared. The latter strains grew more slowly and were less permeable to sucrose and raffinose. They were also more resistant to multiple hydrophilic antibiotics than an isogenic strain containing the 40 kDa protein and were less permeable to penicillin G and chloramphenicol. We conclude that the 40 kDa outer-membrane protein functions as a porin in H. influenzae.

Ca2+ was accumulated by right-side-out membrane vesicles of Bacillus subtilis following imposition of a diffusion potential, inside-negative, owing to K+-efflux via valinomycin. Uptake was dependent on the magnitude of the membrane potential. This voltage-dependent Ca2+ uptake was inhibited by Ca2+ channel blockers such as nitrendipine, verapamil and LaCl3, and was competitively inhibited by Ba2+ and Sr2+. The system showed saturation kinetics with an apparent K m for Ca2+ of about 250 μm. Proteins responsible for the voltage-dependent Ca2+ uptake were partially purified by preparative isoelectric focusing in a Sepharose bed. A fraction at pH 5·28–5·33 contained the activity. The characteristics of Ca2+ uptake in reconstituted proteoliposomes were the same as those in membrane vesicles (sensitive to Ca2+ channel blockers; inhibited by Ba2+ and Sr2+). In addition, uptake was not influenced by a pH gradient imposed on the vesicles. The apparent K m for Ca2+ in the reconstituted system was about 260 μm. The specific activity was increased about 50-fold by purification with isoelectric focusing.

SUMMARY: The properties of an N-acetylmuramidase previously isolated from PL-1 phage lysates of Lactobacillus casei were studied in more detail. The enzyme differed from hen egg white lysozyme in its substrate specificity spectrum, in its physical and chemical nature and in other properties. The cell wall lytic action showed narrow specificity. The enzyme was most active against the phage's host. It was composed of a single polypeptide chain of M r about 37000 as estimated by gel filtration, SDS-PAGE and amino acid analysis. The circular dichroic spectrum showed that the peptide backbone of the enzyme molecule most probably had a mainly random structure. The protein was rich in neutral amino acids, and the enzyme had an isoelectric point of pH 5·0. The N-terminal sequence was determined up to 27 amino acid residues, showing alanine as the N-terminus; the C-terminus was a tyrosine residue.

SUMMARY: Streptococcus mutans strain AHT (serotype g) secretes at least two glucosyltransferases with different pI values. A novel glucosyltransferase with a pI of 5·8 was purified 244-fold from the ammonium sulphate fraction by DEAE-cellulose chromatography, FPLC (Mono Q column, Pharmacia) and hydrophobic chromatography. The enzyme preparation gave a single protein band on analysis by both PAGE and SDS-PAGE, and did not form multiple protein bands detectable by IEF. The M r was estimated to be about 130000 by SDS-PAGE and about 135000 by ultracentrifugal analysis. The apparent K m value and pH optimum of the enzyme were 3·9 ± 0·2 mm (mean ± sd) and about 4·7, respectively. The enzyme synthesized water-soluble glucan from sucrose, and the glucan consisted of over 90 mol% 1,6-α-d-glucosidic linkages. The enzyme activity was not stimulated by primer dextran. Anti-enzyme serum produced a single precipitin band with the purified enzyme preparation, whereas it did not react with either of the other two known glucosyltransferases.

SUMMARY: A facultatively methylotrophic Mycobacterium was isolated from Cleveland Harbor, Ohio, USA. The isolate, designated ID-Y, used a wide range of carbon and energy sources including methane and several other hydrocarbons. It displayed a growth cycle from rod-shaped exponential-phase cells, with many cell pairs exhibiting V-formation, to cocco-bacillary stationary-phase cells. A fixation technique involving glutaraldehyde/alcian blue resulted in the observation of a three-layered cell wall. Isolate ID-Y has an ultrastructure similar to that of other mycobacteria, particularly Mycobacterium phlei and Mycobacterium flavum, which is presently classified as a Xanthobacter species.

A collection of 655 thermosensitive mutants of Bacillus subtilis 168, obtained by indirect selection, was screened for those lysing at the non-permissive temperature. Thirty-three mutations thus identified were distributed by transformation into eight linkage groups designated lssA to lssH. The distribution was non-random. With the exception of group A, all groups were small, suggesting that mutations identified in each of them may map in one gene only. Linkage groups identified here were mapped in four different regions of the B. subtilis chromosome and their positions relative to reference markers were the following: (i) aroI-lssA-dal-purB; (ii) metC-lssB-lssC-furA-pyrE-cysC-lssD; (iii) lssF-gtaA-lssG-hisA-lssH-cysB; and (iv) cysA-lssE-dnaC-purA. Kinetics of N-acetyl-D-[1-14C]glucosamine incorporation revealed that groups A, B, C, D and F are deficient in peptidoglycan synthesis at the restrictive temperature. In group G, anomalies at the cell wall level were suggested by incorporation and growth curves. It appears that in almost all known cases, thermosensitive lysis mutations in B. subtilis either affect genes involved in peptidoglycan synthesis or lead, more or less directly, to induction of prophages.

The nucleotide sequence of the DNA fragment containing the streptomycin 6-phosphotransferase (streptomycin 6-kinase) gene from the streptomycin-producer Streptomyces griseus strain HUT 6037 was determined. Analysis of the sequence revealed an open reading frame which could encode 325 amino acid residues. A biased codon usage pattern, reflecting the high G + C composition (approximately 74%) of Streptomyces DNA, was observed in the gene.

Sau3A fragments of Clostridium thermocellum (NCIB 10682) DNA were ligated into the BamHI site of pBR322 and expressed in Escherichia coli HB101 and a Lac− mutant thereof. Twenty-eight clones with carboxymethylcellulase (CMCase) activity were selected from two libraries by means of the Congo Red plate assay. Restriction enzyme analysis indicated that the CMCase+ clones contained a total of 13 unique DNA inserts. Hybridization of recombinant plasmids with chromosomal DNA confirmed the physical maps in all but one case and was further used to demonstrate the absence of homology between the HindIII restriction fragments of similar size which occurred in many of the clones. Without exception, CMCase+ E. coli clones expressed endoglucanase activity, but differed with respect to the amount and nature of the enzyme activity produced; additionally, some clones had exoglucanase activty which, in at least one case, was not attributable to the production of a second enzyme. For a few selected clones, the partially purified CMCase was analysed by electrophoresis. A temperature profile characteristic of a thermostable enzyme was demonstrated for the endoglucanase of one of the most active clones. Based on the evidence presented here, it is probable that the 13 unique DNA fragments described do not contain any of the C. thermocellum endoglucanase genes previously cloned.

Phages coding for production of Vero cytotoxins VT1 or VT2 in strains of Escherichia coli serotype O157. H7 or O157. H− were morphologically indistinguishable. Their genome size and restriction enzyme digests of the phage DNA were similar. These phages were clearly different in these respects from a VT1-encoding phage isolated from a strain of E. coli O26. H11 (H19). However the VT1 region cloned from the phage originating in the E. coli O157. H7 strain was identical to the VT1 region previously cloned from the phage carried by H19. Sequences encoding VT2 that were cloned from the phage in E. coli O157. H− have been mapped and the VT2 region identified by transposon insertion. The cloned regions coding for VT1 or VT2 production had no similarities in the presence of restriction enzyme sites over a distance of about 2 kb, and two VT1-specific probes spanning a region of about 1·4 kb did not hybridize under stringent conditions with cloned VT2 DNA. A 2 kb HincII fragment contained the VT2 genes but hybridized to VT1-encoding phages and recombinant plasmids via flanking phage DNA. A 0·85 kb AvaI-PstI fragment was a specific probe for VT2 sequences and did not hybridize under stringent conditions to phages or plasmid recombinants encoding VT1.

SUMMARY: Seven phages were fairly susceptible in vitro to the lethal effect of acidified whey, more so than the enteropathogenic Escherichia coli strains on which they were active. The low acidity that prevailed in the abomasum contents of calves shortly after a milk feed had little harmful effect on orally administered organisms of these phages; they flooded into the small intestine. The high acidity that prevailed later was lethal to orally administered phage organisms; few entered the small intestine. The lethal effect could be counteracted by giving CaCO3 in the feed. Low concentrations of phage-neutralizing antibodies were found in some serum samples from human beings, cattle and pigs. Antibodies to one of the seven phages were common in the human samples and antibodies to another, phage B44/1, were common in the cattle and pig samples and in bovine colostrum. Phage B44/1 antibodies in a sample of colostral whey were destroyed at pH 3·5 or less. Giving colostrum containing phage B44/1 antibodies with CaCO3 to a calf greatly reduced the numbers of orally administered phage B44/1 organisms in its alimentary tract. Antibodies to another phage were induced in the serum of a calf suffering from E. coli diarrhoea by treating it with that phage. The phages were as susceptible as the E. coli strains to the lethal action of formaldehyde and sodium hypochlorite. In contrast to the E. coli strains, they were almost completely resistant to phenol and chloroxylenol. The in vitro virulence of 21 phages varied according to the temperature at which tests were performed. The majority were almost avirulent at 20 °C and 43 °C and most virulent at 37 °C. Experiments in calves suggested that their body temperatures were sufficiently high to adversely influence the virulence of some phages.

SUMMARY: Strains of Klebsiella pneumoniae of serotype K21 are frequently involved in outbreaks of nosocomial infections. The type strain of Klebsiella pneumoniae K21 (which we have renamed K21a) produces capsular polysaccharide that contains mannose, galactose and glucuronic acid in the ratio 2:2:1. In contrast, all eight of the randomly chosen isolates of Klebsiella pneumoniae that were initially typed as K21 were shown by paper chromatography and NMR spectroscopy to produce a different capsular polysaccharide. We have designated this new polysaccharide K21b. The K21b capsular material appears to have a closely similar immunodominant side chain to K21a. However, K21b polysaccharide has two molecules of rhamnose in the polysaccharide backbone replacing the two molecules of mannose found in the K21a capsule. Our results suggest that the K21b Klebsiella serotype may be more frequently distributed than the classical K21a type.