Rapid identification ofRhizobium species based on cellular fatty acid analysis

As understanding of the evolutionary relationships between strains and species of root nodule bacteria increases the need for a rapid identification method that correlates well with phylogenetic relationships is clear. We have examined 123 strains ofRhizobium: R. fredii (19),R. galegae (20),R. leguminosarum (22),R. loti (17),R. meliloti (21), andR. tropici (18) and six unknowns. All strains were grown on modified tryptone yeast-extract (TY) agar, as log phase cultures, scraped from the agar, lysed, and the released fatty acids derivatized to their corresponding methyl esters. The methyl esters were analysed by gas-chromatography using the MIDI/Hewlett-Packard Microbial Identification System. All species studied contained 16:0, 17:0, 18:0 and 19cyclo_w9C fatty acids but onlyR loti andR tropici produced 12:0 3 OH,13:0 iso 3 OH,18:1_w9C and 15:0 iso 3 OH,17:0 iso 3 OH and 20:2_w6,9C fatty acids respectively. Principal component analysis was used to show that strains could be divided into clusters corresponding to the six species. Fatty acid profiles for each species were developed and these correctly identified at least 95% of the strains belonging to each species. A dendrogram is presented showing the relationships betweenRhizobium species based on fatty acid composition. The data base was used to identify unknown soil isolates as strains ofRhizobium lacking a symbiotic plasmid and a bacterium capable of expressing a symbiotic plasmid fromR. leguminosarum asSphingobacterium spiritovorum.     

Comparison of techniques for measuring plasmid stability in yeasts

Summary Three techniques for measuring plasmid stability in yeasts are described and evaluated. The yeast used was aKluyveromyces lactis strain which was transformed with a plasmid, pCR1, to enable production of heterologous α-amylase. The techniques were based on plate counts on a selective antibiotic-containing medium and a non-selective medium, and on clearing zones on starch-supplemented plates for α-amylase detection. The plate ratio and clearing zones methods gave comparable results while the transfer colony method estimated much lower plasmid stabilities.     

Relaxation of reproductive suppression in non-breeding female naked mole-rats, Heterocephalus glaber

Ninety-four non-reproductive female naked mole-rats, from seven colonies, were studied in terms of vaginal perforation, vaginal smears and urinary concentrations of oestradiol-17β and progesterone in relation to the time of parturition of the breeding female, the queen. The study concentrated mainly on the period from nine days prepartum to 13 days postpartum of 12 births. Sixty-eight percent ( n = 253) of the non-reproductive females had detectable urinary concentrations of oestradiol-17β and many of these had perforated vaginas throughout the study period. These females showed a significantly increased urinary concentration of oestradiol six days prior to parturition of the queen. In females with undetectable concentrations of oestradiol-17β, the proportion with perforated vaginas increased from six days prepartum (54%) to reach a peak on the day of parturition (92%) of the queen. Urinary progesterone-concentrations were 0.7nmol/mmol creatinine at some stage in the study period in 90% of the females and scattered short peaks or spikes were experienced by all these females, but without synchronization between the females in a colony and without any detectable correlation with the time of parturition of the queen. Maximal concentrations in some females were comparable to the values in cycling breeding females during the luteal phase, but were of a much shorter duration than in breeding females. Vaginal smears did not show clear cyclic patterns.     

The colony structure and reproductive biology of the afrotropical Mashona mole-rat, Cryptomys darlingi

Cryptomys darlingi occurs in the mesic Miombo woodland of north-eastem Zimbabwe. It occurs in colonies of up to nine individuals, in which reproduction is limited to one of the largest males and the largest female in the colony.
Reproduction and details of colony size and number of breeding animals in a colony are described for five complete field-captured colonies.
In captivity, mating is not confined to a particular season, and up to three litters of pups are orn per annum. The reproductive female initiates the pre-copulatory behaviour. The gestation lengti is 56–61 days ( n = 2 ). The new-born pups are altricial and litter size is small = 1.7 ± 0.5 ( n = 6). In this case, the pups first left the nest 10 days after birth, began to eat solids when 14 days old, and were fully weaned at five weeks. They began to spar with each other when 36–40 days old, but did not disperse and were incorporated into the colony. This suggests that the Mashona mole-rat colonies are composed of a founding pair and at least three successive litters of pups.     

Expression and secretion of wheat α-amylase in Kluyveromyces lactis

The plasmid pCR1 has been constructed to express a wheat -amylase enzyme in Kluyveromyces lactis strains. The contruct is based on the vector pCXJ-kan1, which has been derived from pDK1, a native plasmid of K. lactis var. drosophilarum containing the essential regions for plasmid replication and stability. Contruct pCR1 produces an -amylase by DNA isolated from a wheat cDNA clone and is controlled by a Saccharomyces cerevisia PGK promoter. Correspondence to: C. Russell     

Histopathological studies ofSporothrix schenckii-inoculated mice

Defense mechanisms againstSporothrix schenckii were studied using mouse models. After an intracutaneous injection of the yeast form ofS. schenckii to the dorsal skin of the congenitally athymic nude and normal heterozygote littermate mice, nodules were formed. They regressed and disappeared in 10 weeks in the case of normal mice. On the other hand, nodules and then ulceration developed progressively in nude mice until all animals expired by dissemination of microorganisms at the 11th week of inoculation. Histopathologically the migrated cells were similar in both the normal and the nude mice, particularly during the early phase (within 24 h), with infiltration by PMNs being predominant. Fragmentation ofS. schenckii commenced early during the 12–24 h stage of inoculation in the normal mice, while such fragmentation was scarce in nude mice even though numerous PMNs accumulated. Microscopic observations in the early stages (within 24 h of inoculation) suggested that the lack of killing activity by PMNs in nude mice contributes more to the impaired defense than the lack of macrophage activation by T-cells.     

Antibody raised against extracellular proteinases ofSporothrix schenckii inS. schenkii inoculated hairless mice

Sporothrix schenckii produces two extracellular proteinases, namely proteinase I and II. Proteinase I is a serine proteinase, inhibited by chymostatin, while proteinase II is an aspartic proteinase, inhibited by pepstatin. Studies on substrate specificity and the effect of proteinase inhibitors on cell growth suggest an important role for these proteinases in terms of fungal invasion and growth. There has, however, been no evidence presented demonstrating thatS. schenckii produces 2 extracellular proteinases in vivo. In order to substantiate the in vivo production of proteinases and to attempt a preliminary serodiagnosis of sporotrichosis, serum antibodies against 2 proteinases were assayed usingS. schenckii inoculated hairless mice. Subsequent to an intracutaneous injection ofS. schenckii to the mouse skin, nodules spontaneously formed and disappeared for a period of 4 weeks. Histopathological examination results were in accordance with the microscopic observations. Micro-organisms disappeared during the fourth week. Serum antibody titers against purified proteinases I and II were measured weekly, using enzyme-linked immunosorbent assay (EIA). As a result, the time course of the antibody titers to both proteinases I and II were parallel to that of macroscopic and microscopic observations in an experimental mouse sporotrichosis model. These results suggest thatS. schenckii produces both proteinases I and II in vivo. Moreover, the detection of antibodies against these proteinases can contribute to a serodiagnosis of sporotrichosis.     

Gastric colonization withCandida albicans

This is the first report on the isolation ofCryptococcus neoformans from pigeon droppings in China and their serotypes.C. neoformans colonies which produced brown colonies on caffeic acid-cornmeal agar were found in Twenty-five out of thirty-six samples of pigeon droppings. Fifty-one colonies randomly picked from the positive samples were identified asC. neoformans by a commercially available kit for carbon source assimilation test and Christensen's urea agar. Forty (78%) out of the 51 strains were serotyped as A and 11 (22%) as AD. At the same time, seventeen out of nineteen clinical isolates were serotyped as A and 2 as B. There are three findings in our results. One is that onlyC. neoformans var.neoformans strains could be isolated from pigeon droppings, although the varietygattii strains were found in the clinical isolates obtained in the same geographic site in China. The second is that serotype A strains were most frequently seen in natural and clinical materials in the southeast part of China, and serotype AD strains were isolated in pigeon droppings but not in clinical materials. The third is that the coexistence of serotype A and AD cells ofC. neoformans strains in same samples of pigeon droppings were observed.     

Two bacteria causing farmer's lung: Fine structure ofThermoactinomyces vulgaris andSaccharopolyspora rectivirgula

Larval cuticle ofHelicoverpa (Heliothis)zea and yeast extract added to a minimal medium (MM) induced germination of conidia ofNomuraea rileyi whereas sterile distilled water or MM alone did not. Yeast extract increased mycelial yield, but when cuticle was added, mycelial yield significantly decreased. Proteases and chitinases ofN. rileyi were only expressed when cuticle was added to the MM.This article reports the results of research only. Mention of a proprietary product in this paper does not constitute a recommendation for use by US Department of Agriculture.     

Isogenic P-fimbrial deletion mutants of pyelonephritogenic Escherichia coli: the role of α Gal(1–4)β Gal binding in virulence of a wild-type strain

Escherichia coli strains causing acute pyelonephritis often express multiple fimbrial types and haemolysin, which may contribute to their ability to adhere to, and interact with, kidney epithelial cells. Strain CFT073, a pap⁺, sfa⁺, pil⁺, hly⁺ pyelonephritis strain, previously established as virulent in the CBA mouse model of ascending urinary tract infection and cytotoxic for cultured human renal epithelial cells, was selected for construction of isogenic strains. From a gene bank of this strain, two distinct copies of the pap operon were isolated. The two P-fimbrial determinants were sub-cloned into pCVD442, a positive selection suicide vector containing the sacB gene of Bacillus subtilis. Deletion mutations were introduced into each of the two constructs, within papEFG of one operon and papDEFG of the other. Suicide vectors carrying pap deletions were mobilized from E. coli SM10 lambda pir into CFT073 (Nal^R) and cointegrates were passaged on non-selective medium. The first pap mutation was identified by screening a Southern blot of DNA from sucrose-resistant colonies using a papEFG probe. This mutant retained the MRHA⁺ phenotype since a second functional copy of pap was still present. A double pap-deletion mutant, UPEC76, confirmed by Southern blotting, was unable to agglutinate human type O erythrocytes or α Gal(1–4)β Gal-coated latex beads. CBA mice (N =100) were challenged transurethrally with 10⁵, 10⁶, 10⁷, or 10⁹ cfu of strains CFT073 or UPEC76. After one week, quantitative cultures of urine, bladder, and kidney were done and histologic changes were examined. No substantive differences in organism concentration or histological findings between parent and mutant were detected in urine, bladder, or kidney at any challenge concentration. We conclude that adherence by P fimbriae of uro-pathogenic E. coli strain CFT073 plays only a subtle role in the development of acute pyelonephritis in the CBA mouse model.