Robert B. Hespell

A phylogenetic grouping of the bacteroides, cytophagas, and certain flavobacteria

Summary A variety of species of Bacteroides, Flavobacterium, Cytophaga and various of their relatives have been characterized by the method of oligonucleotide cataloging. Except for certain misclassified strains, these organisms form a phylogenetically coherent and major cluster of eubacteria, a eubacterial “phylum”. Within the grouping the bacteroides form a separate subgroup, which has defined internal structure. The cytophagas and flavobacteria, however, group together in what appears to be a rather ancient phylogenetic unit. Although a grouping of the anaerobic bacteroides with the aerobic flavobacteria and cytophagas is not suggested by any known phenotypic similarities, it can be rationalized through the existence of an unnamed isolate (Pl-12fs of K. O. Stetter) which manifests a phenotype in ways intermediate between the two, and which occupies a phylogenetically intermediate position as well.

The phylogeny of the spirochetes

Summary A number of species of Spirochaeta, Treponema, Leptospira and Borrelia have been characterized by the method of ribosomal RNA oligonucleotide cataloging, in order to determine their phylogenetic relationships. The group so formed is phylogenetically very deep, with a major division occurring between the leptospiras on the one side and the remaining spirochetes on the other. At a comparably deep level the greater group also encompasses the recently recognized group of obligate anaerobic halophiles. The main cluster within the greater group comprises three sublines: (1) that containing the bulk of the spirochetes and treponemes examined, (2) that represented by B. hermsii, and (3) that represented by T. hyodysenteriae. The first of these in turn divides into two main subclusters, represented by S. halophila on the one hand and S. stenostrepta on the other.

Trimethylamine and methylamine as growth substrates for rumen bacteria andMethanosarcina barkeri

Trimethylamine and methylamine were found to be used as methanogenic substrates byMethanosarcina barkeri or by bacteria found in low dilutions of rumen contents. When these substrates were used as the only added carbon and nitrogen source, up to 80% of the theoretical amount of methane production was obtained.Methanosarcina were enumerated from rumen contents at 105–106 bacteria/ml. Pure cultures of the various major rumen bacterial species, includingMethanobacterium ruminantium strain M1, were not able to utilize these substrates as energy and/or nitrogen sources. It is suggested that, in the rumen, trimethylamine and methylamine are primarily degraded byMethanosarcina, resulting in release of ammonia which then can be utilized by other rumen bacteria.

Phylogenetic relationships among various helical bacteria

Helical bacteria from the generaSpirillum, Oceanospirillum, Aquaspirillum, andAzospirillum—as well asSerpens flexibilis—were characterized by oligonucleotide cataloging of 16S rRNA in order to establish their phylogenetic relationships to one another and to Gramnegative bacteria in general. The various genera of helical bacteria are not specifically related to one another (to the exclusion of nonhelical bacteria) and, where tested, the individual genera as presently constituted are not phylogenetically coherent (with the possible exception ofOceanospirillum, which may form a deep grouping).

The Origin and Phylogeny of the Bdellovibrios

Summary Five strains of bdellovibrios, Bd. bacteriovorus 109J, 109D, Bd. starrii A3-12, Bd. stolpii , and Bd. BM4, have been characterized by the method of oligonucleotide cataloging. The first three are very closely related to one another. The last two are related to each other as well, but a specific relationship between these two groups could not be detected with certainty. However, all bdellovibrios are related specifically to the major bacterial group defined by the sulfate reducing bacteria and the myxobacteria.

Attempts to grow bdellovibrios micurgically-injected into animal cells

Incubation in buffer of Bdellovibrio bacteriovorus 109J, B. stolpii UKi2, or B. starrii A3.12 with washed eucaryotic animal cells (mouse liver, hamster kidney, or bovine mammary gland) resulted in neither attachment nor growth of the bdellovibrios. When cells of these bdellovibrio strains were incubated with erythrocyte suspensions (bovine or rabbit) a very low level of bdellovibrio attachment and penetration occurred, but no growth could be detected. Using micurgical procedures, bdellovibrios were injected into the perivetelline space or the cytoplasm of rabbit ova. After 18–24h incubation, neither a significant loss nor increase of injected, intracellular bdellovibrios was observed. Limited axenic growth of bdellovibrios (109J or UKi2) occurred in media containing rabbit ova extracts and dilute nutrient broth. It is concluded that eucaryotic rabbit ova do not provide a suitable environment for intracellular bdellovibrio growth.

Survival ofMegasphaera elsdenii during starvation

Batch- and continuous-cultured cell suspensions of the anaerobic ruminal bacteriumMegasphaera elsdenii strain T-81 were subjected to total nutrient starvation, during which time changes in cell viability, cell composition, and endogenous fermentation acids were monitored. The populations exhibited poor survival capabilities with a 50% survival time of 9–13 h. The primary substrates used for endogenous metabolism appeared to be cellular RNA, carbohydrate, and possibly protein. The types and amounts of major fermentation acids (acetic, butyric, caproic) released from starving cells varied depending upon initial growth conditions and starvation time. The data suggest that growth conditions affect cell composition and have important roles in survival ofM. elsdenii.

Effects of nucleic acid compounds on viability and cell composition of Bdellovibrio bacteriovorus during starvation

The effects of various exogenous nucleic acid compounds on the viability and cell composition of Bdellovibrio bacteriovorus starved in buffer were measured. In decreasing order of effectiveness, these compounds were found to decrease the rate of loss of viability and the loss of cell carbon, cell ribonculeic acid, and cell protein: glutamate > ribonucleoside monophosphates > ribonucleosides > deoxyribonucleoside monophosphates. Similar sparing effects were not observed with nucleic acid bases, deoxyribonucleosides, ribose, ribose-5-phosphate, deoxyribose, and deoxyribose-5-phosphate. Appreciable increases in the respiration rate over the endogenous rate did not occur when cell suspensions were incubated with individual or mixtures of nucleic acid compounds. Formation of 14CO2 by cell suspensions incubated with carbon 14-labeled nucleic acid compounds indicated ribonucleosides and ribonucleoside monophosphates were respired and to a small extent, were incorporated into cell material of non-growing cells. The respired 14CO2 was derived mainly from the ribose portion of these molecules. No respired 14CO2 or incorporated carbon 14 was found with bdellovibrios incubated with other nucleic acid compounds tested, including free ribose. During growth of B. bacteriovorus on Escherichia coli in the presence of exogenous UL-14C-ribonucleoside monophosphates, 10–16% of the radioactivity was in the respired CO2 and of the radioactivity incorporated into the bdellovibrios, only 40 to 50% resided in the cell nucleic acids. However, during growth on 14C-adenine,-uracil, or-thymidine labeled E. coli, only trace amounts of 14CO2 were found and 90% or more of the incorporated radioactivity was in the bdellovibrio nucleic acids. It is concluded that bdellovibrio can use ribonucleoside monophosphates during growth and starvation as biosynthetic precursors for synthesis of both nucleic acids and other cell materials as well as catabolizing the ribose portion for energy purposes.