A new species of Neisseria from iguanid lizards, Neisseria iguanae sp. nov.

A new species of the genus Neisseria is proposed, Neisseria iguanae sp. nov. The organism is associated with septic lesions in the common iguana ( Iguana iguana ) and the rhinoceros iguana ( Cyclura cornuta ) but is also a commensal. It resembles N. animalis and N. canis phenotypically but is distinguished from these by exhibiting pronounced tetrad arrangement and alpha haemolysis and by fermenting gluconate.     

Identification of oral Neisseria species of animals

Ninety-seven strains of presumptive Neisseria spp. obtained from the dental plaque of a wide variety of animals and 21 strains from culture collections were compared by physiological tests, enzyme electrophoresis and isoelectric focusing of proteins. Three physiological groups based on the fermentation of maltose and production of extracellular polysaccharide were established. The ability of strains within these groups to reduce nitrate and nitrite differed. The electrophoretic mobility of dehydrogenases and isoelectric focusing patterns of proteins were not, however, characteristic of species or physiological groups. It is difficult, therefore, to identify new isolates of Neisseria because the criteria for describing species overlap. A rapid spot test was devised to distinguish Branhamella catarrhalis from other Neisseria by the absence of glucose-6-phosphate dehydrogenase.     

Selective isolation of Neisseria sicca from the human oral cavity on eosin methylene blue agar.

Strains of Neisseria sicca and N. mucosa, but not N. perflava, N. subflava, N. flava, or N. flavescens were found to grow on eosin methylene blue agar. The distribution of N. sicca on the tongue dorsum, the gingival crevice area, and the coronal surfaces of teeth of humans was determined using this medium. N. sicca averaged about 5% of the total cultivable organisms of the tongues of 14 subjects examined, but it was present in only trace quantities in dental plaque on the coronal surfaces of teeth or in the gingival crevice area.     

A snapshot of a pathogenic bacterium mid-evolution: Neisseria meningitidis is becoming a nitric oxide-tolerant aerobe

Members of the Neisseria genus typically display the ability to carry out denitrification of nitrite to nitrous oxide as an alternative to oxygen respiration when oxygen is depleted. The key enzymes nitrite and nitric oxide reductase are found across the Neisseria genus. Within Neisseria meningitidis, however, a number of research groups have found that a significant proportion of strains lack a functional nitrite reductase. It appears that N. meningitidis is on an evolutionary trajectory towards loss of the capacity to reduce nitrite. In the present paper, I propose that N. meningitidis is evolving to become a nitric oxide-tolerant aerobe in order to occupy an oxygen-rich niche close to host tissue (and hence oxygen perfusion). Other features of the genomic and functional specialization of N. meningitidis, such as possession of a polysaccharide capsule and various acquired reactive oxygen species-resistance mechanisms, support this proposition. The importance of oxygen availability more generally is discussed with reference to recent findings with other mucosal pathogens.     

Effect of Glutamate on Exogenous Citrate Catabolism of Neisseria meningitidis and of Other Species of Neisseria

Resting cell suspensions of Neisseria meningitidis group B (strain 2091) do not catabolize citrate as the sole substrate to an appreciable degree. When another substrate, such as glutamate, is also present to furnish energy for transport, citrate metabolism is greatly stimulated. Within limits, the amount of CO(2) produced from citrate is proportional to the amount of glutamate added. When the cells are disrupted, citrate is degraded at a rapid rate and the stimulatory effect of glutamate is completely eliminated. Pronounced stimulation of citrate metabolism by glutamate was demonstrated in 12 of 13 strains of N. meningitidis tested and only 1 of 6 strains of N. lactamicus. The remaining strains of N. lactamicus and one each of N. gonorrhoeae, N. flavescens, and N. flava did not utilize significant amounts of citrate in the absence or presence of glutamate. N. catarrhalis shared with Mima polymorpha and Moraxella glucidolytica a capability to catabolize citrate at a rapid rate without added glutamate. It is concluded that tests of glutamate-stimulated citrate metabolism may contribute to species characterization in the genus Neisseria.     

Preparation of Cell Walls and Protoplasm of Neisseria with the Ribi Cell Fractionator

The varied pressures required for disruption of Neisseria gonorrhoeae and other species of Neisseria when the Sorvall-Ribi refrigerated cell fractionator is used in the preparation of cell walls and cellular protoplasm are reported. Optimal disruption pressure for the gonococcus was considerably less than that required for other members of the genus Neisseria. Pressures varied from 8,000 psi for N. gonorrhoeae F62, colony type 4, to 22,000 psi for the nonpathogenic Neisseria-N. sicca, N. flava, and N. catarrhalis. Representative electron photomicrographs are shown.     

Deoxyribonucleic Acid Homologies Among Species of the Genus Neisseria

Eleven aerobic species of Neisseria, a Mima sp., and a Herellea sp. were tested for deoxyribonucleic acid (DNA) homology in direct hybridization experiments. DNA labeled with either (14)C or (32)P was prepared from five species of Neisseria. Unlabeled DNA from the various microorganisms was immobilized on membrane filters, which, after pretreatment, were incubated with labeled DNA (4,000 counts per min per filter) for 14 hr at 67 C. The measure of relatedness was expressed as the relative percentage of direct binding compared to that obtained with homologous DNA. All serological types of N. meningitidis, including the newly proposed types, were homologous to the standard strain of N. meningitidis with one possible exception, type Z. The genus Neisseria is heterogeneous in nature, forming at least three distinct groups: first, N. meningitidis and N. gonorrhoeae; second, N. perflava, N. subflava, N. sicca, N. flavescens, and N. flava; third, N. catarrhalis and N. caviae. Mima and Herellea species show no significant homology with the Neisseria.     

Neisseria lactamicus sp. n., a Lactose-fermenting Species Resembling Neisseria meningitidis

The biochemical and serological characteristics of lactose-utilizing strains of Neisseria were determined. These organisms were found in the nasopharynx of man and grew well on Thayer-Martin Selective Medium. They were compared with N. meningitidis to ascertain whether they were variants of this species. Differences between the lactose-using strains and the recognized species of Neisseria were considered significant enough to warrant designation of a new species, Neisseria lactamicus. This group has not been widely recognized as being separate from N. meningitidis; therefore, the normal incidence and clinical significance of these organisms has not been fully established. These organisms are oxidase-positive and positive for beta-D-galactosidase activity; they demonstrate fermentation in King Oxidation-Fermentation Medium; and they produce acid from only glucose, lactose, and maltose, of the 27 substrates incorporated in Cystine Trypticase Agar. Individual strains vary in their ability to grow on Nutrient Agar at both 25 and 37 C and in their pigmentation on Loeffler Medium. Results indicated that these organisms are serologically distinct from the N. meningitidis serogroups. Only 34 of 116 strains of N. lactamicus were smooth and could be tested by slide agglutination. None of the 34 could be grouped as N. meningitidis group A, B, C, D, X, Y, or Z. Thirty-one of these strains could, however, be specifically grouped with antisera prepared with N. lactamicus strains. Cross absorptions confirmed that N. lactamicus is serologically distinguishable from N. meningitidis.     

Interspecies recombination between the penA genes of Neisseria meningitidis and commensal Neisseria species during the emergence of penicillin resistance in N. meningitidis: natural events and laboratory simulation.

The penicillin-binding protein 2 genes (penA) of penicillin-resistant Neisseria meningitidis have a mosaic structure that has arisen by the introduction of regions from the penA genes of Neisseria flavescens or Neisseria cinerea. Chromosomal DNA from both N. cinerea and N. flavescens could transform a penicillin-susceptible isolate of N. meningitidis to increased resistance to penicillin. With N. flavescens DNA, transformation to resistance was accompanied by the introduction of the N. flavescens penA gene, providing a laboratory demonstration of the interspecies recombinational events that we believe underlie the development of penicillin resistance in many meningococci in nature. Surprisingly, with N. cinerea DNA, the penicillin-resistant transformants did not obtain the N. cinerea penA gene. However, the region of the penA gene derived from N. cinerea in N. meningitidis K196 contained an extra codon (Asp-345A) which was not found in any of the four N. cinerea isolates that we examined and which is known to result in a decrease in the affinity of PBP 2 in gonococci.     

Neisseria gonorrhoeae: evaluation of some methods used for carbohydrate utilization.

A total of 989 Neisseria gonorrhoeae cultures were examined for acid production from dextrose in seven different carbohydrate media. Four of these media were found to be quite accurate for this purpose. Some pitfalls in the preparation and usage of cystine trypticase agar carbohydrate media are discussed. The term 'fermentation' of carbohydrates by Neisseria species is inaccurate and responsible for some N. gonorrhoeae false-dextrose-negative results.     

Characterization of the mitogenic activity elicited by Neisseria gonorrhoeae ribosomal fractions.

Ribosomal preparations from Neisseria gonorrhoeae types 1 and 4 were examined for their in vitro stimulation of mouse splenocytes to determine the ribosomal moiety or contaminant responsible for the immunoproliferative activity. In immunodiffusion tests with homologous rabbit antiserum, crude 70S ribosomes formed four precipitin bands while the purified 30S and 50S subunits showed one major line. The same antiserum reacted with lysed N. gonorrhoeae and Neisseria meningitidis A cells but no precipitation occurred with Escherichia coli cells purified N. gonorrhoeae lipopolysaccharide (LPS). No membrane or LPS contaminant was detected in the purified 30S and 50S preparations. All the ribosomal preparations from virulent and non-virulent N. gonorrhoeae consistently stimulated the murine splenocytes. The mitogenic activity of the 30S and 50S ribosomal preparation was destroyed by treatment with trypsin but only slightly decreased by ribonuclease. It is suggested that the lymphoproliferative response elicited by gonococcal ribosomes is triggered by the protein moiety of the 30S or 50S subunits.     

Bioinformatic analysis of outer membrane proteome of Neisseria meningitidis and Neisseria lactamica.

Two-dimensional electrophoresis (isoelectric focusing/SDS-PAGE) and Western-blotting techniques were used to analyze and compare common and/or specific outer-membrane proteins and antigens from Neisseria meningitidis and Neisseria lactamica. Bioinformatic image analyses of proteome and immunoproteome maps indicated the presence of numerous proteins and several antigens shared by N. meningitidis and N. lactamica, although the inter-strain variation in the maps was of similar magnitude to the inter-species variation, and digital comparison of the maps did not reveal proteins found to be identical by MALDI-TOF fingerprinting analysis. PorA and RmpM, two relevant outer-membrane antigens, manifested as various spots at several different positions. While some of these were common to all the strains analyzed, others were exclusive to N. meningitidis and their electrophoretic mobilities were different than expected. One such spot, with a molecular mass of 19 kDa, may be the C-terminal fragment of RmpM (RmpM-Cter). The results demonstrate that computer-driven analysis based exclusively on spot positions in the proteome or immunoproteome maps is not a reliable approach to predict the identity of proteins or antigens; rather, other identification techniques are necessary to obtain accurate comparisons.     

Alteration of epithelial cell transferrin-iron homeostasis by Neisseria meningitidis and Neisseria gonorrhoeae

Iron is an essential element for nearly all organisms. In mammals, iron is transported to body tissues by the serum glycoprotein transferrin. Transferrin-iron is internalized by binding to specific receptors followed by endocytosis. In vitro , Neisseria meningitidis and Neisseria gonorrhoeae can use iron from a variety of iron-containing compounds, including human transferrin. In vivo , transferrin is an important source of iron for N. gonorrhoeae : a mutant that is unable to bind and use transferrin-iron is unable to colonize the urethra of men or initiate disease at this site. As pathogenic Neisseria and its human host derive much of their iron from transferrin, we reasoned that a competition may exist between microbe and host epithelial cells for transferrin-iron at certain stages of infection. We therefore tested the hypothesis that N. meningitidis and N. gonorrhoeae may actively interfere with host transferrin-iron metabolism. We report that Neisseria-infected human epithelial cells have reduced levels of transferrin receptor messenger RNA and cycling transferrin receptors. The ability of infected cells to internalize transferrin receptor is also reduced. Finally, the relative distribution of surface and cycling transferrin receptors is altered in an infected cell. We conclude that Neisseria infection alters epithelial cell transferrin-iron homeostasis at multiple levels.     

Type IV prepilin peptidase gene of Neisseria gonorrhoeae MS11: presence of a related gene in other piliated and nonpiliated Neisseria strains.

The assembly of type IV pili in Neisseria gonorrhoeae is a complex process likely to require the products of many genes. One of these is the enzyme prepilin peptidase, which cleaves and then N methylates the precursor pilin subunits prior to their assembly into pili. We have used a PCR amplification strategy to clone the N. gonorrhoeae prepilin peptidase gene, pilDNg. A single copy of the gene is shown to be present in the chromosome. Its product promotes correct cleavage of the gonococcal prepillin in Escherichia coli cells carrying both the prepilin peptidase gene and the pilin structural gene. PilDNg also cleaves prePulG, a type IV pilin-like protein of Klebsiella oxytoca. Moreover, PilDNg complements a mutation in the gene coding for the prepilin peptidase-like protein of K. oxytoca, pulO, partially restoring PulG-PulO-dependent extracellular secretion of the enzyme pullulanase. Finally, we show that genes homologous to pilDNg are present and expressed in a variety of species in the genus Neisseria, including some commensal strains.     

Growth pattern and cell division in Neisseria gonorrhoeae.

The gram-negative coccus Neisseria gonorrhoeae was found to grow regularly in at least two dimensions. Growth proceeded at a linear rate sequentially in each dimension. Growth in the second dimension (former width) was initiated slightly before the pole-division plane distance equalled the cell width. Penicillin treatment localized presumptive growth zones to the existing septum region. It was suggested that new growth zones were always formed perpendicular to the longitudinal axis created in the incipient daughter cells of a dividing coccus. Neither penicillin nor nalidixic acid induced filaments of N. gonorrhoeae. Such structures could nevertheless be formed in the rod-shaped species Neisseria elongata. N. gonorrhoeae divides by septation; however, complete septal structures with separated cytoplasms were rather infrequent. It is proposed that N. gonorrhoeae be regarded as a short rod which always extends parallel to the actual longitudinal axis and which never undergoes a rod-sphere-rod transition.     

Site-specific insertion of IS1301 and distribution in Neisseria meningitidis strains.

The insertion element IS1301 has been shown to mediate capsule phase variation in Neisseria meningitidis found in N. serogroup B by reversible insertional inactivation of the siaA gene. We have determined the target site specificity of this element by cloning and sequencing the insertion sites of 12 identical IS1301 copies found in N. meningitidis B1940. A target consensus core of 5'-AYTAG-3' was identified, with the central TA being duplicated following insertion. Additional features around the target sites, including extended palindromic symmetry, stem-loop formation, and the high incidence of AT tracts, indicate that other factors, such as DNA secondary structure, are involved in target recognition. The left inverted repeat of an IS1016-like element acts as a hot spot for insertion, with one insertion element combination located upstream of their gene. According to further sequence analysis, we were able to place IS1301 in the IS5 subgroup within the IS4 family of elements. A survey of 135 Neisseria strains indicated the presence of IS1301 in 27.9 to 33.3% of N. meningitides serogroup B, C, and W135 strains and in 86.7% of serogroup Y strains. IS1301 did not occur in serogroup A strains, in Neisseria gonorrhoeae, or in apathogenic Neisseria spp.     

Conjugal transfer of beta-lactamase-producing plasmids of Neisseria gonorrhoeae to Neisseria meningitidis

Twenty clinical isolates of beta-lactamase-producing Neisseria gonorrhoeae from Japanese sources were studied to define their ability to serve as donors for their plasmids in conjugation with Neisseria meningitidis. These twenty strains of N. gonorrhoeae harbored the 4.5-megadalton (Mdal) beta-lactamase-producing plasmids and the 24.5-Mdal conjugative plasmids. We found that only three of twenty N. gonorrhoeae strains showed a detectable conjugation frequency (greater than 10(-5)) with N. meningitidis as the recipient although all strains were capable of mobilizing beta-lactamase-producing plasmids to N. gonorrhoeae and to Escherichia coli. The 4.5-Mdal beta-lactamase-producing plasmid was maintained in N. meningitidis, but the large 24.5-Mdal conjugative plasmid has not been found in N. meningitidis transconjugants.     

Identification and characterization of specific sequences encoding pathogenicity associated proteins in the genome of commensal Neisseria species

Abstract The distribution of distinct sequences in pathogenic and commensal Neisseria species was investigated systematically by dot blot analysis. Probes representing the genes of Rmp, pilin and IgA1 protease were found to hybridize exclusively to the chromosomal DNA of the pathogenic species, Neisseria gonorrhoeae and/or Neisseria meningitidis . In contrast, specific sequences for the genes of the porin protein Por and the opacity protein (Opa) were also detected in a panel of commensal Neisseria species such as N. lactamica, N. subflava, N, flava, N. mucosa and N. sicca . Using opa -specific oligonucleotides as probes in chromosomal blots, the genomes of the commensal Neisseria species show a totally reduced repertoire of cross-hybridizing loci compared to the complex opa gene family of N. gonorrhoeae . DNA sequence analysis of one opa -related gene derived from N. flava and N. sicca , respectively, revealed a large degree of homology with previously described gonococcal and meningococcal genes e.g., a typical repetitive sequence in the leader peptide and the distribution of the hypervariable and conserved regions. This observation, together with the finding, that the gene is constitutively transcribed, leads to the assumption that some of the commensal Neisseria species may have the potential for the expression of a protein harboring similar functions as the Opa proteins in pathogenic Neisseriae .     

Multicolonization of human nasopharynx due to Neisseria spp.

The colonization due to Neisseria spp. in the nasopharynx of forty healthy adults was studied by using a selective medium that allows the differentiation of Neisseria species and inhibits the rest of pharyngeal microbiota. The medium detected a variety of colonial morphology types and some metabolic characteristics of the isolates. We demonstrated the multicolonization by several Neisseria spp. in the same individual, and we isolated several strains of the same species, after analysis by pulsed-field gel electrophoresis (PFGE) patterns obtained from the different colonial types previously identified as the same species. The forty adults studied were colonized by 112 forms of Neisseria spp., and twelve colonization patterns were obtained: one species (45%), two (45%), three (7.5%) and four (2.5%). N. perflava-N. sicca, either alone or in combination with other species was the most frequent isolate (92.5%). The analysis of PFGE patterns obtained from different colonial types revealed the multicolonization by several strains of the same species in some individuals. This fact was found in N. perflava-N. sicca (50%) and N. mucosa (2.5%).     

HmbR outer membrane receptors of pathogenic Neisseria spp.: iron-regulated, hemoglobin-binding proteins with a high level of primary structure conservation.

We have recently cloned and characterized the hemoglobin receptor gene from Neisseria meningitidis serogroup C. N. meningitidis cells expressing HmbR protein were able to bind biotinylated hemoglobin, and the binding was specifically inhibited by unlabeled hemoglobin and not heme. The HmbR-mediated hemoglobin binding activity of N. meningitidis cells was shown to be iron regulated. The presence of hemoglobin but not heme in the growth medium stimulated HmbR-mediated hemoglobin binding activity. The efficiency of utilization of different hemoglobins by the HmbR-expressing N. meningitidis cells was shown to be species specific; human hemoglobin was the best source of iron, followed by horse, rat, turkey, dog, mouse, and sheep hemoglobins, The phenotypic characterization of HmbR mutants of some clinical strains of N. meningitidis suggested the existence of two unrelated hemoglobin receptors. The HmbR-unrelated hemoglobin receptor was shown to be identical to Hpu, the hemoglobin-haptoglobin receptor of N. meningitidis. The Hpu-dependent hemoglobin utilization system was not able to distinguish between different sources of hemoglobin; all animal hemoglobins were utilized equally well. HmbR-like genes are also present in N. meningitidis serogroups A and B, Neisseria gonorrhoeae MS11 and FA19, Neisseria perflava, and Neisseria polysaccharea. The hemoglobin receptor genes from N. meningitidis serogroups A and B and N. gonorrhoeae MS11 were cloned, and their nucleotide sequences were determined. The nucleotide sequence identity ranged between 86.5% (for N. meningitidis serogroup B hmbR and MS11 hmbR) and 93.4% (for N. meningitidis serogroup B hmbR and N. meningitidis serogroup C hmbR). The deduced amino acid sequences of these neisserial hemoglobin receptors were also highly related, with overall 84.7% conserved amino acid residues. A stop codon was found in the hmbR gene of N. gonorrhoeae MS11. This strain was still able to use hemoglobin and hemoglobin-haptoglobin complexes as iron sources, indicating that some gonococci may express only the HmbR-independent hemoglobin utilization system.