Method for determining the specific growth rate of distinct microbial populations in a non-homogeneous system

The present invention pertains to some molecular biology-based method and kit for quantifying the specific growth rate (or cell regeneration period ) of distinct microbial populations. The method and kit can be used to examine mixed culture samples which were subjected to chloramphenicol or other protein synthesis inhibitors for defined times. In a preferred embodiment, the technique of the invention (also referred to herein as FISH-RiboSyn) is an in situ procedure that uses fluorescence in situ hybridization (FISH) with probes that target: (1) the 5′ or 3′ end of precursor 16S rRNA; or (2) the inside area of both precursor 16S rRNA and mature 16S rRNA. Images can be captured for a defined exposure period and the average fluorescent intensity for human cells could be determined. The rate of increase of the whole cell fluorescent intensity is used to ascertain the specific growth rate. The procedure of the invention can be attractive for quickly measuring the specific growth rate (or cell doubling time) of distinct microbial populations inside a mixed culture in sectors such as environmental systems (water and wastewater treatment systems), bioremediation (optimization of conditions for microbial growth), public health (identification of fast growing infectious microbes), and homeland security (identification of fast growing bioterrorism agents).


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Before the 1970s, the phylogeny of the prokaryotes was predicated on primitive comparisons of morphology and pattern of substrate use and has been mostly ignored due to the supposed simplicity of the organisms. Carl Woese used a different strategyto tackle prokaryotic phylogeny. He concentrated on sequence comparisons of the ribosome, a biomolecule found in all life forms. The ribosome is a vital macromolecule that’s involved in the translation of messenger RNA into proteins. Woese arguedthat since protein synthesis is a vital role for life, the ribosome couldn’t withstand major sequence changes or life would stop. Then he targeted one molecule, the 16S rRNA of both prokaryotes and the analogous 18S rRNA for eukaryotes, and didcomparisons by sequence analysis (Woese, C. R. and G. E. Fox Proc. Natl. Acad. Sci. USA, 1977, 74:5088-5090). A new phylogeny of life was detected and to his surprise (and other biologists), the older phylogeny of eukaryotes and prokaryotes wasdiscarded for a three-kingdom version that included bacteria, archaea, and eucarya (shown in FIG. 1). As time passes, most biologists have recognized this paradigm change. So far, 35 bacteria phyla and 18 archaea phyla were identified, despite just having 30cultivatable representatives for both (Hugenholtz, P. Genome Biol, 2002, 3(2):0003). With the discovery of a robust bacterial phylogeny from Woese, molecular biology-based methods have slowly replaced traditional techniques in the analysis of microbialpopulations in environmental samples (Woese, C. R. et al.. Proc. Natl. Acad. Sci. USA, 1990, 87:4576-4579). All these molecular biology based methods rely on the 16S rRNA, the biomolecule used by Woese to determine the phylogeny of bacteria andarchaea. Over the past twenty decades, molecular biology software development has progressed from determining community structure to community function.

Specific microbial populations have a exceptional sequence touch inside the 16S or 18S rRNA. Norman Pace recognized that specific microbial populations have trademark sequences over the 16S or 18S rRNA which can be targeted at molecular biologybased methods. Pace’s group was the first to show using fluorescence in situ hybridizations using an oligonucleotide probe that is complementary to those signature sequences (DeLong, E. F. et al.. Science, 1989, 243(4896):1360-3). With thisapproach, they were able to identify and enumerate microbes inside a mixed culture sample in different phylogenetic levels. Nowadays, probes and their hybridization characteristics for specific microbial populations are available throughconvenient websites (Loy, A. et al.. Nucleic Acids Res, 2003, 31(1):514-516). By way of instance, the arrangement, hybridization conditions, and other characteristics of an oligonucleotide probe which targets that the 16S rRNA of the genus Nitrospira (ProbeBaseaccession number pB-00627) are as follows: specificity: Nitrospira spp.; target molecule: 16S rRNA; position: 447-464; sequence: 5′-GGTTTCCCGTTCCATCTT-3′ (SEQ ID NO:1); length: 18 nt; G+C content: 50%; Tm: 48. degree. C.; delta Gs: .DELTA. G.sub.1:-22.03; .DELTA. G.sub.2: 1.41; .DELTA. G.sub.12: -21.96; MW: 5406 g/mol; formamide: 30 percent; (Schramm, A. et al.. Appl. Environ. Microbiol., 1998, 64:3480-3485; information offered by ProbeBase, an internet database of probes in the Department of MicrobialEcology, University of Vienna).

Molecular biology-based methods have now replaced classical methods in the analysis of microbes. Considering that Pace’s demonstration of FISH, molecular biology-based methods have been developed to investigate microbial populations in combined cultures, suchas bioreactors and environmental samples. As shown by FIG. 2, three types of molecular biology-based techniques have been developed to identify, enumerate, and determine the function of specific microbial populations. A fourth class of molecular biologybased techniques provides a measure of this diversity. Every one these molecular biology-based methods draw on the order information of their 16S rRNA.

The analysis of the microbiology of mixed culture samples involves determining the identity and abundance of microbes present (microbial community structure) and their role in the mixed culture sample (microbial community function).Traditionally, light microscopy or culture-based methods were used to characterize the microbial arrangement of blended culture samples. More recently, new tools which draw on molecular biology and a brand new perspective of the phylogeny of life have been developed toidentity bacteria and determine their purpose.

Molecular biology tools have been used to determine community structure and function. The first wave of molecular biology tools identify and enumerate specific microbial populations in environmental systems. Recently, Amann et al. (Amann, al.. FEMS Microbiology Ecology, 1998, 25:205-215) reviewed molecular biology based methods for identifying and enumerating bacterial inhabitants and these are summarized below. For particular microbial populations where the 16S rRNA sequenceinformation can be obtained, tools are available to identify human cells in situ (fluorescence in situ hybridizations or FISH) (DeLong, E. F. et al.. Science, 1989, 243(4896):1360-3) or provide estimates of abundance for a microbial population ex situ(membrane hybridizations). For uncharacterized samples, researchers use DNA amplification by polymerase chain reaction (PCR) that targets large phylogenetic groups along with conventional cloning methods to identify the different kinds of microbespresent. Ultimately, fingerprinting methods such as terminal restriction length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE) characterize the diversity and evenness of environmental samples (Liu, W. T. Water Science andTechnology, 1998, 37(4-5): 417-422; Kaewpipat, K. and C. P. Grady, Jr.. Water Sci Technol, 2002, 46(1-2):19-27; Kreuzinger, N. et al.. Water Sci Technol, 2003, 47(11):165-72).

The second wave of molecular biology tools decided the purpose of specific microbial populations in situ or ex situ. FISH is combined with microautoradiography (FISH-MAR) to supply a method that identifies microbes that metabolize specificcompounds. With FISH-MAR, ecological samples are exposed to radio-labeled substrates. In some cases, the rate of substrate uptake has been reported (Nielsen, J. L. et al.. Environ Microbiol, 2003, 5(3):202-11). FISH-MAR is a difficult method tomaster, which restricts its acceptance as another wave instrument. An ex situ method named Isotope Array is based on precisely the same principle as FISH-MAR, however, membrane hybridizations are used to identify the dominant microbial population linked to substrate uptake(Adamczyk, J. et al.. Appl Environ Microbiol, 2003, 69(11):6875-87).

Molecular biology tools for examining the development activity of microbial communities in environmental samples are being used. Three approaches are used for determining the growth activity of the parasitic members in biologicalreactor systems. The easiest strategy entails detecting and enumerating the germs which are only able to perform certain metabolic capabilities. In cases like this, a very simple identification and enumeration from the methods employed for microbial structureanalysis are required. The next approach determines the abundance of enzymes or mRNA present in a sample that’s particular for an enzyme in the particular metabolic pathway of interest. The identification of these microbes containing these genes or mRNA isnot always possible, because these biomolecules are not phylogenetic markers and are present at reduced cellular levels. The next approach determines whether the microbes of interest have been growing. With this strategy, the measurement of the rRNA existing inthe cells is required. Membrane hybridizations are used by researchers as proof that a bacterial population is active when their relative 16S rRNA levels grow. Detection of increased ribosome synthesis has been utilized to ascertain whenbacterial populations or cells of a bacterial inhabitants are continuously growing. These approaches and others involving genetically modified organisms are reviewed (Molin, S, and M. Givskov Environmental Microbiology, 1999, 1(5):383-391).

For the last 50 years, scientists have been measuring the specific growth rate of pure cultures by using spectrophotometers (watch FIGS. 3A and 3B). Over time, the optical density is measured to get a specified wavelength and compared to a sterile thatcontains sterile broth media. With a very simple spreadsheet, the specific growth rate of the culture is determined by evaluation of the rate of growth of the optical density.

The specific rate of ribosome synthesis (or ribosome doubling time) is equal to the specific growth rate (or cell doubling time) of this civilization. During log gain, cells are growing at a constant specific growth rate, which also means theyhave a specified and continuous doubling time. In the same way, the ribosome doubling time needs to be equal to the cell doubling time, which can be portrayed in FIG. 4.

Throughout the 1960’s, researchers first reported the macromolecular composition of pure cultures was determined by the development rate (Maaloe, O. and N. O. Kjeldgaard,”Control of Macromolecular Synthesis; a research of DNA, RNA, and proteinsynthesis in germs” 1966, New York: W. A. Benjamin, p. 284). The association between the macromolecular composition and expansion period of E. coli strain B/r is revealed in Table I (Bremer, H. and P. P. Dennis,”Modulation of chemical composition andother parameters of the cell by growth rate” in Escherichia coli and Salmonella, F. C. Neidhardt, et al., Editors; 1996, ASM Press: Washington, D.C.). Two basic descriptors of ribosome synthesis, rRNA transcription and cellular ribosome amounts, are alsoincluded. The rRNA transcription is reported as the portion of total transcription.

TABLE-US-00001 TABLE 1 Comparison of certain growth rate, rRNA transcription, and macromolecular composition of E. coli strain B/r. Particular Growth rRNA Ribosomes Rate transcription per mobile Composition% hr.sup.-1 percent — RNA DNA Protein 0.6 356,800 14 5 68 2.5 73 72,000 24 2 52

An approximately 10-fold growth in ribosome level is detected when E. coli increases its specific growth rate from 0.6 hr.sup.-1 to 2.5 hr.sup.-1. During rapid expansion, over 50 percent of the total RNA generated in E. coli is ribosomal RNA (rRNA),which is notable given that there are only 14 promoters connected with the seven rrn operons compared to 2,000 totalpromoters available (Gourse, R. L. and M. Nomura,”Prokaryotic rRNA gene saying, in Ribosomal RNA: structure, evolution,processing, and function in protein biosynthesis” R. A. Zimmermann and A. E. Dahlberg, Editors. 1996, CRC Press, Inc.: Boca Raton. p. 373-394). The most significant macromolecule fraction for all growth rates is protein. As the growth rate rises, the RNAcontent increases and protein content decreases. This results from the increase of ribosome amounts or stable RNA. Bremer and Dennis (Bremer, H. and P. P. Dennis,”Modulation of chemical composition and other parameters of the cell by growth rate” inEscherichia coli and Salmonella, F. C. Neidhardt, et al., Editors; 1996, ASM Press: Washington, D.C.) developed a growth equation for E. coli which was a function of constant ribosome concentration (amount of ribosomes per protein) and action (proteinsynthesis rate per ribosome).

Some researchers have used fluorescence in situ hybridizations with probes that target the ribosomes in cells and noted that faster growing cells possess greater levels of ribosomes according to fluorescent intensity (DeLong, E. F. et al.. Science,1989, 243(4896):1360-3; Poulsen, L. K. et al.. Appl Environ Microbiol, 1993, 59(5):1354-60). However, this approach was lost as a way of measuring the particular growth rate (or mobile regeneration period ), since cells maintain high levels of ribosomesduring stationary stage which would be thought of as rapidly expanding cells.

Central to microbial growth is ribosome synthesis, the creation of functional ribosomes. Presently, the ribosome synthesis version of Escherichia coli is the most complete, best understood, and recognized to characterize ribosome synthesis forBacteria. A fundamental review of E. coli ribosome synthesis is offered below, nevertheless several comprehensive reviews of E. coli ribosome synthesis can be found (Gourse, R. L. and M. Nomura,”Prokaryotic rRNA gene saying, in Ribosomal RNA. Structure, development, processing, and function in protein biosynthesis” R. A. Zimmermann and A. E. Dahlberg, Editors. 1996, CRC Press, Inc.: Boca Raton. P. 373-394; Jemiolo, D. K.”Performance of Prokaryotic ribosomal RNA” at Ribosomal RNA: structure, development,processing, and function in protein biosynthesis, R. A. Zimmermann and A. E. Dahlberg, Editors; 1996, CRC Press, Inc.: Boca Raton, p. 453-468; Srivastava, A. K. and D. Schlessinger Annual Review of Microbiology, 1990, 44:105-129). A schematic ofribosome synthesis in bacteria is revealed in FIG. 5. Expression of the rrn operon creates a polycistronic transcript composed of the 3 rRNAs: 5S, 16S, and 23S. Two processing measures are required to produce mature rRNAs for ribosome assembly. Inthe primary processing measure, RNaseIII cleaves the polycistronic transcript resulting in 3 precursor rRNAs: precursor 5S (pre5S), precursor 16S (pre16S), and precursor 23S (pre23S). A secondary processing measure removes unnecessary RNA from the 5′ and3′ endings of this precursor rRNAs before ribosome assembly. This secondary processing measure is slower compared to the primary processing step, which causes an intracellular pool of precursor rRNAs.

Chloramphenicol interrupts ribosome synthesis. As shown in FIG. 6 and FIG. 7, chloramphenicol inhibits the secondary processing of precursor 16S rRNA, but does not inhibit the production of precursor 16S rRNA (Tomlins, R. I. and Z. J. Ordal JBacteriol, 1971, 107(1):134-42). Cangelosi and Brabant (Cangelosi, G. A. and W. H. Brabant Journal of Bacteriology, 1997, 179(14):4457-4463) employed a reverse osmosis method to measure the level of precursor 16S rRNA in cells of E. coli which wereexposed to chloramphenicol. Their results suggested a marked difference in the speed of the buildup of the pre16S rRNA in growing and non-growing cells which were exposed to chloramphenicol. Chloramphenicol treated E. coli cells have been also reported tohave considerably higher level of pre16S rRNA than normally observed for LB civilizations (Licht, T. R. et al.. Environmental Microbiology, 1999, 1(1):23-32).

FIG. 7 is a simplified example of a cell in log growth phase that’s subjected to chloramphenicol. Within this figure, the initial degree of pre16S rRNA is zero than the degree of 16S rRNA (80,000), which signifies ribosomes. After exposure tochloramphenicol, the degree of 16S rRNA stays constant, whereas the pre16S rRNA increases to 40,000 after 15 minutes and 80,000 after 30 minutes. For non-growing cells (e.g., in static phase) subjected to chloramphenicol, the degree of pre16S rRNA and16S rRNA will remain constant.


U.S. Pat.

Nos. 5,770,373; 5,726,021; and 5,712,095, that are all incorporated by reference herein in its entirety, describe methods for identifying chloramphenicol-resistant strains of mycobacteria, and the normal reaction of ribosomesynthesis into chloramphenicol. U.S. Patent Application Publication No. 200400772242, that is incorporated herein by reference in its entirety, describes a way of detecting, enumerating and/or identifying microorganisms in a sample. U.S. PatentApplication Publication No. 20060105339, that is incorporated herein by reference in its entirety, describes a way of quantifying the rates of replication and death of parasitic infectious agents in an infected host organism. A molecularbiology-based method which measures the specific growth rate (or cell doubling time) of different microbial populations in a mixed culture hasn’t previously been reported.

The analysis of microbial populations through using molecular biology-based methods has been a boon for researchers in the areas of environmental science and technology, microbial ecology, drug discovery, public health, homelandsecurity, etc.. An molecular biology-based tool which measures the particular growth rate of distinct microbial populations would be of fantastic interest to scientists and engineers that reveal an interest in deciding how quickly microbes are now growing. Industriesthat may benefit include, but aren’t limited to, environmental systems (water and wastewater treatment methods ), bioremediation (optimisation of conditions for microbial growth), public health (identification of fast growing infectious germs ), andhomeland safety (identification of fast growing bioterrorism agents).

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